Synovial cell protein

ABSTRACT

The present invention discloses a novel protein called Synoviolin and a gene that encodes it. This protein is expressed specifically by synovial tissue and also accompanies the presence of an auto-antibody that recognizes this protein in rheumatoid arthritis (RA) patients. The protein according to the present invention and its antibody can be expected to be used as specific diagnostic markers for RA. In addition, the gene or protein according to the present invention may be used to permit the screening of drugs to treat RA. Moreover, the present invention provides synoviolin gene transgenic animals. The transgenic animals according to the present invention can be used as RA model animals in the development of pharmaceuticals to treat RA.

TECHNICAL FIELD

The present invention relates to a novel protein pertaining torheumatoid arthritis (RA), a polynucleotide that encodes this proteinand applications for said protein or polynucleotide. More specifically,it relates to a novel protein that can be expected to serve as aspecific diagnostic marker for RA. In addition, it also relates to anovel gene that provides new approaches to the development of drugs forthe treatment of RA.

BACKGROUND ART

RA is a chronic inflammatory disease of the entire body whereinhyperplasia is seen in the synovial tissue of joints. Synovial cells arefibroblastoid cells that form the one to six epithelioid layers of thesynovial membranes of joints, and are thought to supply proteoglycan andhyaluronic acid to the synovial fluid. Hyperplasia of the synovialtissue is seen in the joints of RA patients along with the resultingsymptoms of multilayer structures and infiltration of synovial cellsinto other tissue caused thereby. In addition, the blood serum of an RApatient contains autoantibodies to the Fc domain of its own IgG.Accordingly, this is thought to be an autoimmune disease, but its causehas yet to be elucidated.

The aforementioned presence of autoantibodies that recognize self-IgGhas been long utilized as a characteristic diagnostic indicator of RA.Autoantibody detection kits containing modified human IgG as the maincomponent have recently become commercially available. This autoantibodyis also called the RA factor. The diagnosis of RA based on the detectionof the RA factor has problems with respect to specificity to the diseaseand that the relationship to the cause is unclear since the system bywhich antibodies occur has not been elucidated.

When the pathology of RA is examined from the two aspects of that of thevarious immune reactions in the body and that of a hyperplastic diseaseof the joint synovial membrane accompanying bone disruption, muchresearch has been performed regarding the former immune reactions andthe molecular mechanism thereof is about to be clarified. However,regarding study of the latter joint synovial cells, even though this isa principal aspect of RA, even their cytobiological characteristics haveto be clarified at present. Elucidating the molecular mechanism behindthe onset and progress of RA and other chronic and intractable diseasesis indispensable for the diagnosis, prevention and cure of the diseases.Moreover, in the current situation in which the aging of society doesnot show signs of halting, elucidating the pathology of the agingdisease RA is an important problem from a societal standpoint also.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to provide a novel protein thatprovides new approaches to the diagnosis and treatment of RA and a novelgene that encodes this protein. The protein provided by the presentinvention and the polynucleotide that encodes it are closely related tothe cause of RA and will provide useful information for diagnosis, andalso lead to the creation of new drugs in the development of treatmenttechniques. Moreover, further objective of the present invention is toprovide transgenic animals wherein the genes that encode said proteinsare expressed, along with knockout animals that lack said gene. Theseanimals will be useful in analyzing the functions of the gene accordingto the present invention and also in the development of RA treatmentmethods and treatment drugs as model animals.

The present inventors used anti-human synovial cell antibodies obtainedusing cultured human synovial cells from RA patients as the immunogen toperform immunoscreening of a cDNA library of synovial cells of RApatients, and thus succeeded in isolating a new gene expressed in thesynovial tissue of RA patients. The protein encoded by this gene wasnamed Synoviolin after the synovial cells which are the tissues in whichthis gene is expressed.

The present inventors have confirmed that the reactivity of anti-humansynovial cell antibodies to about 80-kDa, 140-kDa, and 220-kDa molecularweight fractions of the aforementioned cultured synovial membrane cellsis absorbed by the expression products of the aforementioned synoviolingene. In addition, the present inventors found that these bands and theexpression products of the aforementioned synoviolin gene exhibitreactivity to antibodies present in the blood of RA patients. Moreover,the inventors confirmed that anti-human synovial cell antibodies exhibitstrong reactivity to the synovial tissue of RA patients.

In addition, the present inventors used biochemical linkage experimentsto demonstrate the presence of the Synoviolin ligand (SL), which is anatural ligand of Synoviolin. SL is a protein that the present inventorsfor the first time isolated as a ligand of Synoviolin. However, when anattempt was made to perform a search based on nucleotide sequences forthe DNA that encodes SL, a known gene called S1-5 was found to include acommon nucleotide sequence in the 5′ terminal domain and 3′ terminaldomain. The SL isolated by the present inventors and S1-5 are nearlyidentical not only in the DNA partial sequence but also the size of thegene, the molecular weight of the expression product and other aspects,and thus there is a good probability that they are the same protein.S1-5 [also called “FBNL” (fibrillin-like) or “EFEMP1” (EGF-containingfibrillin-like extracellular matrix protein 1)] has been isolated as agene that is overexpressed in a human diploid fibroblast (Lecka-Czernik,B., et al., Molecular and Cellular Biology (1995) 15:120-128).Structurally, it has an epidermal growth factor (EGF)-like domain thatpromotes DNA synthesis. The structure and nucleic acid synthesispromotion activity (hyperplastic activity) of S1-5 have been found. Inaddition, mutations of S1-5 have recently been reported to be associatedwith Malattia Leventinese (ML) and Doyne honeycomb retinal dystrophy(DHRD) (Stone, E. M., et al., Nature Genetics (1999) 22:199-202), but noassociation with RA was known. In addition, it is needless to say thatthe affinity with Synoviolin is completely new knowledge acquired by thepresent inventors.

Moreover, the present inventors have prepared a transgenic mouse byintroducing the synoviolin gene and a knockout mouse that lacks thesynoviolin gene and observed their phenotypes. When the Synoviolinmolecule is excessively expressed in the mouse, hyperplasia of synovialmembranes in joints and bone and cartilage disruption were found, thusexhibiting symptoms resembling those of rheumatoid arthritis. On theother hand, when the synoviolin gene was completely (homozygously)knocked out, incomplete limb bud and skeletal formation were found inthe mouse during the fetal stage. These phenotypes suggest thecontribution of Synoviolin not only to synovial tissue but also to thegeneration, differentiation, regeneration and metabolism of cartilageand bone tissue. In addition, in the arthropathic lesions of Synoviolinoverexpression mice, metabolism and regeneration are actively induced inthe synovial membrane, cartilage and bone tissue. These results clearlydemonstrate that the Synoviolin molecule contributes to RA and otherforms of arthropathy. Moreover, it has been confirmed that Synoviolinoverexpression mice are useful as arthropathic model animals.

Based on this new knowledge, the present inventors have clarified theutility of Synoviolin and its gene, its antibodies or ligands in medicaltreatment or diagnosis, thereby completing the present invention.Moreover, the present inventors prepared a transgenic animal byintroducing the synoviolin gene, and have demonstrated its utility as adisease model for RA. In addition, the present inventors prepared aknock-in animal by substituting the lacZ gene for the synoviolin gene.The synoviolin gene knock-out and lacZ gene knock-in animal makes itpossible to analyze the effects of the lack of the synoviolin gene andalso allows the activity of the synoviolin gene promoter to be easilydetected by the detection of LacZ (as β-galactosidase activity)expressed by an endogenous promoter of the synoviolin gene. Using thisknock-in animal, it is possible to perform screening for compounds thatregulate the expression of the synoviolin gene. It is thought that itshould be also possible to prevent hyperplasia of synovial membranes andto remit the disease by suppressing the overexpression of the synoviolingene in the joints of rheumatism patients.

The gene discovered by the present inventors is closely related to thehyperplasia of synovial tissue, which is the main component of thedisease of RA, and provides extremely important information fordiagnosis. In addition, in contributing to the hyperplasia of synovialtissue, which is the cause of RA, the present invention's gene, itsexpression product, autoantibodies to the expression product, and alsoligands of the expression product are thought to be material that isindispensable in the explanation of the pathology of RA. In particular,the discovery of autoantibodies that recognize Synoviolin in the bloodof RA patients gives a completely new approach in the diagnosis of RA.In addition, these substances will lead to brand-new approaches in thedevelopment of RA treatment methods also.

In addition, the mutations of S1-5 identified as Synoviolin ligands areassociated with ML and DHRD, and thus it is possible that Synoviolincontributes to these diseases also. Accordingly, Synoviolin may be usedin the diagnosis of these diseases, while compounds that regulate thebinding of Synoviolin ligands to Synoviolin or compounds that act asligands of Synoviolin or the like become candidates as medicines forthese diseases.

In addition, Synoviolin is expressed in undifferentiated mesenchymalcells during development. Accordingly, it is possible to use Synoviolinas a cell marker to isolate undifferentiated mesenchymal cells in a cellsorter or the like. The undifferentiated mesenchymal cells thus isolatedcan be utilized for in vitro tissue regeneration. If the in vitroreconstruction of joints is possible, then this would be useful for thereconstructive medical treatment of not only rheumatoid arthritispatients but also many patients suffering from joint damage.

To wit, the present invention relates to the following Synoviolinprotein, an antibody thereof, a polynucleotide that encodes thisprotein, applications thereof, Synoviolin ligands and theirapplications, along with transgenic animals wherein the expression ofthe synoviolin gene is modified and applications thereof.

[1] A polynucleotide selected from the group consisting of (a) through(e) below:

-   -   (a) a polynucleotide that encodes a protein comprising the amino        acid sequence shown in SEQ ID NO: 2,    -   (b) a polynucleotide comprising a protein coding domain of the        nucleotide sequence shown in SEQ ID NO: 1,    -   (c) a polynucleotide encoding a protein that comprises the amino        acid sequence shown in SEQ ID NO: 2 in which one or more amino        acids are substituted, deleted, inserted and/or added and that        is functionally equivalent to the protein consisting of the        amino acid sequence shown in SEQ ID NO: 2,    -   (d) a polynucleotide that hybridizes under stringent conditions        with a polynucleotide consisting of the nucleotide sequence        shown in SEQ ID NO: 1 and that encodes a protein functionally        equivalent to the protein consisting of the amino acid sequence        shown in SEQ ID NO: 2, and    -   (e) a polynucleotide that comprises a nucleotide sequence having        at least 70% or greater identity to the nucleotide sequence        shown in SEQ ID NO: 1 and that encodes a protein functionally        equivalent to the protein consisting of the amino acid sequence        shown in SEQ ID NO: 2.

[2] A polynucleotide that encodes a partial peptide of a proteinconsisting of the amino acid sequence shown in SEQ ID NO: 2.

[3] A protein or peptide encoded by the polynucleotide according to [1]or [2].

[4] The protein or peptide according to [3] that has at least oneactivity selected from the group consisting of the following (1) through(3):

-   -   (1) binds to antibodies found in the blood of rheumatoid        arthritis patients,    -   (2) binds to the Synoviolin ligand S1-5, and    -   (3) promotes synovial membrane hyperplasia.

[5] A vector into which the polynucleotide according to [1] or [2] isinserted.

[6] A transformed cell that carries the polynucleotide according to [1]or the vector according to [5].

[7] A method of manufacturing the protein or peptide according to [3],said method comprising the steps of culturing the transformed cellaccording to [6] and recovering the expressed protein or peptide fromsaid transformed cell or the culture supernatant.

[8] An antibody that binds to the protein or peptide according to [3].

[9] An immunological analysis reagent for analyzing antibodies thatrecognize the protein or peptide according to [3], said reagentcomprising the protein or peptide according to [3].

[10] The immunological analysis reagent according to [9], wherein thereagent is used to diagnose rheumatoid arthritis or to judgeeffectiveness of treating it.

[11] An immunological analysis reagent for analyzing the proteinaccording to [3], said reagent comprising an antibody that reacts withthe protein or peptide according to [3].

[12] The immunological analysis reagent according to [11], wherein thereagent is used to diagnose rheumatoid arthritis or to judgeeffectiveness of treating it.

[13] The immunological analysis reagent according to [12], wherein theprotein according to [3] is present in synovial cells.

[14] A method of measuring antibodies in a biological specimen, whereinsaid antibodies bind to the protein according to [3] and/or a partialpeptide thereof, said method comprising the following steps of:

-   -   (1) contacting the biological specimen with the protein        according to [3] and/or a partial peptide thereof, and    -   (2) detecting the antibodies that bind to the protein according        to [3] and/or a partial peptide thereof.

[15] A method of measuring the protein according to [3] and/or a partialpeptide thereof in a biological specimen, said method comprising thefollowing steps of:

-   -   (1) contacting the biological specimen with the antibody        according to [8], and    -   (2) detecting the antibody according to [8], wherein said        antibody binds to the protein according to [3] and/or a partial        peptide thereof.

[16] A polynucleotide that comprises at least 15 nucleotides and that iscomplementary to a polynucleotide consisting of the nucleotide sequenceshown in SEQ ID NO: 1 or to a complementary strand thereof.

[17] A method of measuring the polynucleotide according to [1] or [2] ina biological specimen, said method comprising the following steps of:

-   -   (1) contacting the biological specimen with the polynucleotide        according to [16], and    -   (2) detecting the polynucleotide according to [16], wherein said        polynucleotide hybridizes with the polynucleotide according to        [1] or [2].

[18] A kit for measuring the polynucleotide according to [1] or [2],said kit comprising the polynucleotide according to [16].

[19] A method of detecting or isolating cells that express the proteinaccording to [3], said method comprising the step of using, as an index,said protein or the expression of a gene that encodes said protein.

[20] The method according to [19], wherein said cells are rheumatoidsynovial cells.

[21] The method according to [19], wherein said cells areundifferentiated mesenchymal cells.

[22] A reagent for the detection or isolation of cells that express theprotein according to [3], said reagent comprising the antibody accordingto [8].

[23] A method of detecting rheumatoid arthritis, wherein the marker forrheumatoid arthritis is at least one selected from the group consistingof the polynucleotide according to [1], the protein according to [3],the peptide according to [3], antibodies that bind to the proteinaccording to [3], and antibodies that bind to the peptide according to[3], the method comprising the following steps of:

-   -   i) detecting the markers for rheumatoid arthritis present in a        biological specimen from a subject, and    -   ii) associating the results of detection of step i) with        rheumatoid arthritis.

[24] The method according to [23], wherein the biological specimen isblood from a subject and the marker for rheumatoid arthritis comprisesantibodies that bind to the protein according to [3] and/or antibodiesthat bind to the peptide according to [3].

[25] The method according to [23], wherein the biological specimencomprises synovial tissue or synovial cells from a subject and themarker for rheumatoid arthritis comprises the polynucleotide accordingto [1] and/or the protein according to [3].

[26] A method of detecting the binding activity of a test compound tothe protein or peptide according to [3], said method comprising thefollowing steps of:

-   -   a) contacting the test compound with the protein or peptide        according to [3], and    -   b) observing the binding of the test compound to said protein or        peptide.

[27] A method of screening compounds that have the activity of bindingto the protein or peptide according to [3], said method comprising thefollowing steps of:

-   -   a) detecting the binding activity of test compounds to the        protein or peptide according to [3] by the method according to        [26], and    -   b) selecting the test compounds the binding activity of which is        higher than a control.

[28] A method of detecting the activity of blocking the binding of theprotein according to [3] to its ligand, said method comprising thefollowing steps of:

-   -   a) contacting the protein or peptide according to [3] with its        ligand in the presence of a test compound, and    -   b) detecting the ligand and/or test compound that bind to said        protein or peptide.

[29] The method according to [28], wherein the ligand is the Synoviolinligand S1-5.

[30] A method of screening compounds that have the activity of blockingthe binding of the protein according to [3] to its ligand, said methodcomprising the following steps of:

-   -   a) detecting the activity of test compounds to block the binding        of the protein according to [3] to its ligand by the method        according to [28], and    -   b) selecting the test compounds said blocking activity of which        is higher than a control.

[31] A method of detecting the activity of a test compound to regulatesignal transduction via the protein according to [3], said methodcomprising the following steps of:

-   -   a) contacting the test compound with said protein either in the        presence of, or in the absence of, the ligand of said protein,        and    -   b) detecting signal transduction via said protein.

[32] A method of screening compounds that have the activity ofregulating signal transduction via the protein according to [3], saidmethod comprising the following steps of:

-   -   a) detecting the activity of test compounds to regulate signal        transduction via the protein by the method according to [31],        and    -   b) selecting the test compounds said regulation activity of        which is higher than a control.

[33] A method of detecting the activity of regulating the expression ofthe polynucleotide according to [1], said method comprising thefollowing steps of:

-   -   a) culturing cells that express the polynucleotide according to        [1] in the presence of a test compound, and    -   b) measuring the expression level of said polynucleotide.

[34] A method of screening compounds that regulate the expression of thepolynucleotide according to [1], said method comprising the followingsteps of:

-   -   a) detecting the activity of test compounds to regulate the        expression of the polynucleotide according to [1] by the method        according to [33], and    -   b) selecting the test compounds that have a difference in said        activity in comparison to a control.

[35] An agent that stimulates Synoviolin, said agent comprising as anactive ingredient a compound that is obtainable by the screening methodaccording to [27].

[36] An agent that blocks the binding between Synoviolin and Synoviolinligand, said agent comprising as an active ingredient a compound that isobtainable by the screening method according to [30].

[37] An agent that blocks synovial hyperplasia, said agent comprising asan active ingredient a compound that is obtainable by the screeningmethod according to [30] or [32].

[38] A pharmaceutical composition that comprises as an active ingredienta component selected from the group consisting of the polynucleotideaccording to [1] or [2], the protein or peptide according to [3], andthe vector according to [5].

[39] A transgenic non-human vertebrate in which the expression of thepolynucleotide according to [1] or [2] is modified or said modificationis inducible.

[40] The transgenic non-human vertebrate according to [39], wherein thepolynucleotide according to [1] or [2] is exogenously transformed.

[41] The transgenic non-human vertebrate according to [40], wherein saidvertebrate is a rheumatoid arthritis model animal.

[42] A transgenic non-human vertebrate in which the expression of theendogenous polynucleotide according to [1] or [2] is suppressed.

[43] The transgenic non-human vertebrate according to [42], whereinanother gene is knocked in.

[44] A cell derived from the transgenic non-human vertebrate accordingto [40] or [42].

[45] A method of detecting the activity of regulating the activity of anendogenous promoter of the polynucleotide according to [1] or [2], saidmethod comprising the following steps of:

-   -   a) contacting a test compound with an expression system that        expresses a reporter gene under the control of the endogenous        promoter of the polynucleotide according to [1] or [2], and    -   b) measuring the expression level of the reporter gene.

[46] The method according to [45], wherein said expression system is thetransgenic non-human vertebrate according to [43] or cells derived fromthe vertebrate.

[47] A method of screening compounds that regulate the activity of theendogenous promoter of the polynucleotide according to [1] or [2], saidmethod comprising the following steps of:

-   -   a) measuring the activity of test compounds to regulate the        activity of the endogenous promoter of the polynucleotide        according to [1] or [2] by the method according to [45], and    -   b) selecting the test compounds that have a difference in said        activity in comparison to a control.

[48] A pharmaceutical composition for regulating the expression of thepolynucleotide according to [1], said pharmaceutical compositioncomprising as an active ingredient a compound that is obtainable by thescreening method according to [34] or [47].

In addition, the present invention relates to a method of stimulatingSynoviolin comprising a step of administering a compound that isobtainable by the screening method according to [27]. Alternatively, thepresent invention relates to a method of blocking the binding ofSynoviolin to Synoviolin ligand comprising a step of administering acompound that is obtainable by the screening method according to [30].Furthermore, the present invention relates to a method of blockingsynovial hyperplasia comprising a step of administering a compound thatis obtainable by the screening method according to [30] or [32]. Inaddition, the present invention relates to a method of promotingsynovial hyperplasia comprising a step of administering a componentselected from the group consisting of the polynucleotide according to[1] or [2], the protein or peptide according to [3], and the vectoraccording to [5]. Moreover, the present invention relates to a method ofregulating the expression of the polynucleotide according to [1]comprising a step of administering a compound that is obtainable by thescreening method according to [34] or [47].

In addition, the present invention relates to use of a compound that isobtainable by the screening method according to [27] for the manufactureof an agent that stimulates Synoviolin. Alternatively, the presentinvention relates to use of a compound that is obtainable by thescreening method according to [30] for the manufacture of an agent thatblocks the binding between Synoviolin and Synoviolin ligand. Moreover,the present invention relates to use of a compound that is obtainable bythe screening method according to [30] or [32] for the manufacture of anagent that blocks synovial hyperplasia. In addition, the presentinvention relates to use of a component selected from the groupconsisting of the polynucleotide according to [1] or [2], the protein orpeptide according to [3], and the vector according to [5] for themanufacture of an agent that promotes synovial hyperplasia. Moreover,the present invention relates to use of a compound that is obtainable bythe screening method according to [34] or [47] for the manufacture of apharmaceutical composition for regulating the expression of thepolynucleotide according to [1].

The present invention provides a polynucleotide that encodes Synoviolincontaining a protein coding domain of the nucleotide sequence shown inSEQ ID NO: 1. The polynucleotide in the present invention may be eitherDNA or RNA. In addition, it may also include a modified nucleotide. Thepolynucleotide that encodes the Synoviolin according to the presentinvention can be cloned from said synovial cells from a RA patient byknown methods (Nucleic Acid Res. (1988) 16:7583-7600). Specifically, acDNA library is obtained based on mRNA extracted from synovial cellsderived from tissue in which the onset of arthritis has occurred, wherethe tissue is recovered from an RA patient as synovial tissue orcultured cells (Nucleic Acid Research (1988) 16:7583). A probe designedbased on the nucleotide sequence shown in SEQ ID NO: 1 can be used toisolate, from this library, the synoviolin gene by the screening ofclones to which the probe hybridizes.

The present invention also encompasses any polynucleotide that encodes aprotein that is functionally equivalent to the aforementionedSynoviolin. In the present invention, a polynucleotide that encodes aprotein that is functionally equivalent to the Synoviolin is referred toas a polynucleotide functionally equivalent to Synoviolin. First, afunctionally equivalent protein is defined as a protein that isimmunologically equivalent to Synoviolin. To wit, in the presentinvention, a protein functionally equivalent to Synoviolin can be adomain of Synoviolin as long as it reacts with antibodies thatspecifically recognize Synoviolin and are present in the blood serum ofan RA patient. Alternatively, it can also be a fragment of a proteinthat contains this immunologically active domain. Mutants thereof can beeasily selected by a person skilled in the art by screening forfragments of Synoviolin using an RA patient blood serum panel and theblood serum of non-afflicted controls.

A protein functionally equivalent to the Synoviolin according to thepresent invention is defined not only based on immunologicalcharacteristics but also based on the characteristic of binding to SL(S1-5). To wit, the present invention encompasses fragments ofSynoviolin that have affinity to SL (S1-5). Mutants thereof can beeasily selected by a person skilled in the art by screening forcandidate proteins using SL (S1-5). For example, as shown in Examples,the SL (S1-5) discovered by the present inventors demands, for thebinding to Synoviolin, 120 amino acid residues corresponding to numbers1233-1592 in the cDNA of Synoviolin. Accordingly, the protein consistingof the amino acid sequence that constitutes this domain, or the proteinincluding this amino acid sequence constitutes a protein functionallyequivalent to the Synoviolin according to the present invention. Aprotein that can be used as SL may be the S1-5 protein identified byaccession number AAA65590 (nucleotide accession U03877), I38449,NP_(—)061489 (nucleotide accession NM_(—)018894), NP_(—)004096(nucleotide accession NM_(—)004105), or Q12805, or a similar proteinthat binds to the human Synoviolin protein (SEQ ID NO: 2)(Lecka-Czernik, B. et al., Mol. Cell. Biol. 15, 120-128, 1995; Heon, E.et al., Arch. Ophthalmol. 114, 193-198, 1996; Ikegawa, S. et al.,Genomics 35, 590-592, 1996; Katsanis, N. et al., Hum. Genet. 106, 66-72,2000; Giltay, R. et al., Matrix Biol. 18, 469-480, 1999; Stone, E. M. etal., Nat. Genet. 22, 199-202, 1999).

In addition, examples of proteins functionally equivalent to humanSynoviolin include proteins that have the activity of promoting synovialhyperplasia. Transgenic mice into which the human synoviolin gene wasoverexpressed were found to exhibit the swelling of toes thataccompanies arthritis with a significant frequency. Histologically, bonedisruption with synovial hyperplasia and abnormal osteogenesis wereobserved in their toe joints. A protein functionally equivalent to thehuman Synoviolin protein may also be defined based on the activity ofpromoting synovial hyperplasia. The promotion of synovial hyperplasiacan be verified by the creation of transgenic animals, or also by thelocal introduction of genes into joints, or by the expression ofproteins in in vitro cultured synovial cells. The method of obtainingtransgenic animals using the polynucleotide according to the presentinvention is described later.

Examples of proteins that are functionally equivalent to humanSynoviolin include proteins that have activity contributing to theformation of normal bones and the development of limbs. In development,Synoviolin was expressed strongly in the parietal bone, limbs, ears andother regions where bone and cartilage are formed, and in the limbformation stage, strong expression was observed in the apical ectodermalridge (AER) and the anlage of cartilage and bone. Knock-out mouseembryos that have the endogenous synoviolin gene knocked out bytargeting have a short length from the parietal region to the buttocks,and a trend for the formation of the skull and limbs to be premature wasfound. The homozygote exhibited abnormal formation in limb buds, theupper and lower jawbones and ears, leading to fetal death with a highprobability. The synoviolin gene homozygous knock-out mouse exhibitedabnormalities in the formation of limb buds in the fetal stage;formation of cartilage and bone was not found; and the expression ofSynoviolin was found in the limb buds and regions of generation ofcartilage and bone, thus demonstrating that the Synoviolin moleculecontributes to skeletal formation and the development of limbs.

In an analysis using a culture system based on the explant method, theexpression of LacZ in cells derived from the limb buds of a synoviolinknock-out (lacZ gene knock-in) mouse embryo was found only inundifferentiated mesenchymal cells that are thought to be the anlage ofcartilage, bone and limbs. Moreover, by the alkaline phosphatase stain,von Kossa stain or other methods, it was confirmed that the capacity toform bone and cartilage was delayed in homozygous knockout mouse-derivedcells. The contribution to normal bone formation and limb development isthought to be verifiable by the creation of knock-out animals, and also,by using analysis of the expression of marker genes of bone andcartilaginous cells in in vitro culture and analysis of the capacity ofbone formation. In addition, the fact that a certain protein hasactivity contributing to normal bone formation and limb development canalso be confirmed in a knock-out animal or cultured cells in which theexpression of the polynucleotide according to the present invention hasbeen suppressed, by administering a protein encoded by saidpolynucleotide, or by the fact that the lost functions are restored bythe expression of DNA or RNA that encodes said protein.

In addition, the protein functionally equivalent to the Synoviolinaccording to the present invention may also be defined based on thebiochemical activity of Synoviolin. The biochemical activity ofSynoviolin can be defined as tyrosine kinase or ubiquitin ligaseactivity, for example. These biochemical activities are corroborated byvarious motifs discovered in Synoviolin and the results of Examples. Towit, the present invention encompasses fragments of Synoviolin thatmaintain at least one biochemical activity that Synoviolin has. Themethod of confirming the biochemical activity of Synoviolin and thedomains where the respective biochemical activities are kept arespecifically described later.

These proteins that are functionally equivalent to Synoviolin can becombined with other proteins to form fusion proteins. For example, aprotein to which a FLAG tag, HA tag, histidine tag or other additionalamino acid sequence is added but which maintains at least one of theproperties of the aforementioned proteins that are functionallyequivalent to Synoviolin is also included in said functionallyequivalent proteins. Even in the event that the added protein hasactivities different from those of Synoviolin, that fusion protein isincluded in the functionally equivalent proteins according to thepresent invention, as long as it keeps at least one of the functions ofSynoviolin.

Polynucleotides comprising nucleotide sequences that contain mutationsin the aforementioned polynucleotide according to the present inventionmay also be isolated by persons skilled in the art using known methods(Jikken Igaku Bessatsu•Idenshi Kōgaku Handobukku [Experimental Medicine,Supplement—Genetic Engineering Handbook], (1991) pp. 246-251, YodoshaCo., Ltd.). For example, if screening is performed on a librarycontaining similar genes using the nucleotide sequence shown in SEQ IDNO: 1 (or a fragment thereof) as a probe, then it is possible to cloneDNA having a nucleotide sequence with a high degree of homology. As sucha library, it is possible to use one that includes random mutations inthe nucleotide sequences of SEQ ID NO: 1, a cDNA library of synovialtissue derived from non-human species, etc.

Examples of known methods of randomly adding mutations to a givennucleotide sequence include the substitution of base pairs by thenitrous acid treatment of DNA (Proc. Natl. Acad. Sci. USA, (1982)79:7258-7260). With this method, it is possible to introduce the randomsubstitution of base pairs within a specific segment by treating thesegment in which mutations are to be introduced with nitrous acid. Astechniques for inducing intended mutations at arbitrary locations, thereare also the gapped duplex and other methods (Methods in Enzymol. (1987)154:350-367). A circular double-stranded vector into which the gene tobe mutated has been cloned is made into a single strand and hybridizedwith a synthetic oligonucleotide that has a mutation at the targetlocation. Complementary single-stranded DNA derived from a linearizedvector cut by restriction enzymes is annealed to the aforementionedcircular single-stranded vector. The gap between the aforementionedsynthetic nucleotide and the complementary single-stranded DNA is filledwith DNA polymerase and ligation is performed to form a completedouble-stranded circular vector.

The number of modified amino acids is thought to be typically 50 aminoacids or less, preferably 30 amino acids or less, and even morepreferably 5 amino acids or less (e.g., 1 amino acid).

When amino acids are artificially substituted, if they are substitutedfor amino acids with similar properties, the original activity of theprotein is thought to be more easily maintained. The proteins accordingto the present invention include proteins to which conservativesubstitutions are added in the aforementioned amino acid substitution,and functionally equivalent to the human Synoviolin protein (SEQ ID NO:2). Conservative substitutions are thought to be important in the caseof substituting the amino acids in domains that are important to theactivity of the protein, etc. Such conservative substitutions of aminoacids are well known to persons skilled in the art.

Examples of amino acid groups for conservative substitution includebasic amino acids (e.g., lysine, arginine, and histidine), acidic aminoacids (e.g., aspartic acid and glutamic acid), uncharged polar aminoacids (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, and cysteine), nonpolar amino acids (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, andtryptophan), β-branched amino acids (e.g., threonine, valine, andisoleucine), aromatic amino acids (e.g., tyrosine, phenylalanine,tryptophan, and histidine) and others.

In addition, non-conservative substitution is thought to increase theactivity or the like of the protein (e.g., including constitutivelyactive proteins and the like) or decrease same (e.g., including dominantnegatives and the like).

A protein that has the amino acid sequence according to SEQ ID NO: 2 inwhich one or more amino acids are substituted, deleted, inserted and/oradded, and that is functionally equivalent to the protein consisting ofthe amino acid sequence shown in SEQ ID NO: 2, also includes a naturalprotein. The genes of eukaryotes typically have polymorphism as seen inthe interferon gene and the like. Changes in the nucleotide sequencearising due to this polymorphism may include cases where one or moreamino acids are substituted, deleted, inserted and/or added. The presentinvention also encompasses a protein that is naturally present and thatis a protein that has the amino acid sequence according to SEQ ID NO: 2in which one or more amino acids are substituted, deleted, insertedand/or added, and that is functionally equivalent to the proteinconsisting of the amino acid sequence shown in SEQ ID NO: 2.

In fact, the present inventors have cloned the gene according to thepresent invention from a plurality of individuals and, by determiningits nucleotide sequence, confirmed a clone from which one amino acid isdeleted. The present invention encompasses a protein that includes suchmutations in the amino sequence, and a polynucleotide comprising anucleotide sequence that encodes it. The nucleotide sequence of theclone missing one amino acid confirmed by the present inventors is shownin SEQ ID NO: 6, and the amino acid sequence encoded by this nucleotidesequence is shown in SEQ ID NO: 7. The nucleotide sequence of SEQ ID NO:6 lacks the gca corresponding to 1293-1295 in SEQ ID NO: 1. As a result,the amino acid sequence according to SEQ ID NO: 7 lacks the Ala inposition 412 of SEQ ID NO: 2.

Alternatively, in some cases, even if there is a change in thenucleotide sequence due to polymorphism, the amino acid sequence may notchange. Such mutations in the nucleotide sequence are called silentmutations. The present invention also encompasses genes comprising anucleotide sequence that has silent mutations. Polymorphism as referredto herein means that a certain gene has different nucleotide sequencesamong individuals within a group. Polymorphism is unrelated to theratios in which different genes are found.

In addition, methods by which a protein functionally equivalent toSynoviolin is obtained may include a method that utilizes hybridization,for example. To wit, this is a method wherein a polynucleotide orfragment thereof that encodes the Synoviolin according to the presentinvention as shown in SEQ ID NO: 1 is used as a probe and apolynucleotide that can be hybridized therewith is isolated. Ifhybridization is performed under stringent conditions, then apolynucleotide with high homology as the nucleotide sequence isselected, and consequently, the probability of a protein functionallyequivalent to Synoviolin being contained in the protein to be isolatedbecomes higher. A nucleotide sequence with high homology is defined tobe one that is 70% identical or greater, or preferably 90% identical orgreater, for example.

Examples of the stringent conditions include conditions of, for example,hybridization at 6×SSC, 40% formamide and 25° C., and washing at 1×SSCand 55° C. While stringency is affected by the conditions such as saltconcentration, formamide concentration or temperature, it is clear thata person skilled in the art may set these conditions so that therequired stringency is obtained.

By using hybridization, it is possible to isolate a polynucleotide thatencodes a homologue of Synoviolin in non-human animal species, forexample. A homologue of Synoviolin encoded by a polynucleotide that canbe obtained from a mouse, rat, rabbit, pig, goat or other non-humananimal species constitutes a functionally equivalent protein in thepresent invention.

There are no limitations on the source of the polynucleotide of thepresent invention. To wit, it may be obtained from cDNA, genome DNA orsynthesis. In addition, it may include a polynucleotide that has anarbitrary nucleotide sequence based on the degeneracy of genetic code,as long as it can encode the protein according to the present invention.

A protein obtained by introducing mutations into human Synoviolin (SEQID NO: 2) and a protein encoded by a polynucleotide isolated using theaforementioned hybridization technique and the like normally have highhomology to human Synoviolin (SEQ ID NO: 2) in the amino acid sequence.High homology means that the sequence is 30% identical or greater,preferably 50% identical or greater, or more preferably 80% identical orgreater (e.g., 95% or greater). The identity of nucleotide and aminoacid sequences can be determined using a homology search site on theInternet [e.g., at the DNA Data Bank of Japan (DDBJ), the FASTA, BLAST,PSI-BLAST, SSEARCH or other homology searches can be used [e.g., the DNAData Bank of Japan (DDBJ) http web site's homology search (Search andAnalysis) page: www.ddbj.nig.ac.jp/E-mail/homology-j.html] and at theNational Center for Biotechnology Information (NCBI), a search usingBLAST can be performed (e.g., the NCBI home page http web site's BLASTpage: www.ncbi.nlm.nih.gov/BLAST/; Altschul, S. F., et al., J. Mol.Biol. (1990) 215(3):403-410; Altschul, S. F. & Gish, W., Meth. Enzymol.(1996) 266:460-480; Altschul, S. F., et al., Nucleic Acids Res. (1997)25:3389-3402)].

For example, calculation of the identity of the amino acid sequence canbe performed in Advanced BLAST 2.1 by using blastp as the program,setting the Expect value to 10, setting all Filters to OFF, usingBLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gapcost and Lambda ratio to 11, 1 and 0.85, respectively, (default values)and performing a search. The value (%) of the identity can then beobtained (Karlin, S. and S. F. Altschul, Proc. Natl. Acad. Sci. USA(1990) 87:2264-2268; Karlin, S. and S. F. Altschul Proc. Natl. Acad.Sci. USA (1993) 90:5873-5877).

The present invention provides applications of these polynucleotidesbesides the production of proteins. To wit, the present inventionencompasses anti-sense polynucleotides against polynucleotides andportions thereof that encode Synoviolin provided by the presentinvention. The anti-sense polynucleotide preferably has a chain lengthof roughly 15-20 nucleotides in order to block the transcription ofgenes effectively. If Synoviolin supports the abnormal hyperplasia ofsynovial cells, then Synoviolin anti-sense polynucleotide has a majorrole in the treatment of RA. From the standpoint of the control of geneexpression, it is also possible to design not only anti-sensepolynucleotide but a ribozyme also. To wit, it is possible to design aribozyme that recognizes and cuts RNA transcribed from the coding regionof DNA shown in SEQ ID NO: 1.

The present invention also relates to polynucleotides with a chainlength of at least 15 nucleotides which are complementary to thepolynucleotide according to the present invention or a complementarystrand thereof. These polynucleotides are those with a chain length ofpreferably 20 nucleotides or more, more preferably 25 nucleotides ormore, or even more preferably 30 nucleotides or more which arecomplementary to the polynucleotide according to the present inventionor a complementary strand thereof. Herein, a “complementary strand”indicates the other strand corresponding to one strand of adouble-stranded nucleic acid consisting of the base pairs A:T (or U inthe case of RNA) and G:C. In addition, “complementary” is defined not tobe limited to the case where there is a completely complementarysequence in a domain of at least 15 consecutive nucleotides, but alsoincludes nucleotide sequences that have homology in the nucleotidesequence of at least 70%, preferably at least 80%, more preferably 90%and even more preferably 95% or greater. The algorithm used fordetermining homology may be one recited herein. These include, forexample, polynucleotides that hybridize with the aforementionedpolynucleotides according to the present invention and that have a chainlength of at least 15 nucleotides.

Hybridization is preferably specific to the polynucleotides according tothe present invention. Herein, the term “specific” means that, understringent hybridization conditions, significant cross-hybridization doesnot occur with polynucleotides that encode other proteins.

These polynucleotides are useful as probes and primers that permit thedetection and amplification of synoviolin genes. It is preferable thatthe probes and primers according to the present invention have a chainlength of at least roughly 15 mer and have a nucleotide sequence thatcan hybridize to a sequence specific to synoviolin within the nucleotidesequence of SEQ ID NO: 1 so that specific hybridization will be possibleunder a given stringency. It is obvious for a person skilled in the artto design a useful nucleotide sequence for the probe or primer based ona given nucleotide sequence. Using the synoviolin gene-specific probesor primers provided based on the present invention, in situhybridization and PCR of synovial cell sample become possible. BecauseSynoviolin is overexpressed in the synovial tissues of RA patients, anunderstanding of the state of expression in the cells is thought to giveimportant information for understanding RA arthritic symptoms.

Synoviolin, which is the novel protein according to the presentinvention, can be obtained from the synovial tissue of RA patients.Because synovial cells can be cultured in vitro, it is possible torecover Synoviolin from this culture. Specifically, synovial cells areisolated from synovial tissue or the like surgically removed from RApatients in a synovectomy. By culturing the isolated cells, it ispossible to recover synovial cells as adhesive cells (J. Clin. Invest.(1993) 92:186-193). Synoviolin is extracted and purified from therecovered cells by a combination of known protein purificationtechniques.

The present invention encompasses not only human Synoviolin extractedfrom synovial cells but also proteins that are functionally equivalentto Synoviolin. To wit, the protein according to the present inventionmay be produced either artificially or naturally, and encompasses amutant protein that has the amino acid sequence of human Synoviolin (SEQID NO: 2) in which one or more amino acids are substituted, deleted,inserted and/or added and that is functionally equivalent to humanSynoviolin. There is no limitation on the number or locations of aminoacid mutations in these proteins as long as the functions of Synoviolinare preserved.

Fragments of Synoviolin can be obtained by digestion using protease. Inaddition, they can also be obtained by randomly cutting the DNA thatencodes the Synoviolin shown in SEQ ID NO: 1, and inserting them intophage vectors to create a phage library that presents domain peptides.If this library is subjected to immunoscreening with antibodies thatrecognize Synoviolin, it is possible to determine the immunologicallyactive domain. The technique for determining the immunologically activedomain can also be used without modification as a technique fordetermining the domain of binding activity with the ligand. Regardingthe cloned phages, if the nucleotide sequence of the inserted fragmentis determined, then the amino acid sequence of the active domain canalso be clarified.

The protein according to the present invention or a protein functionallyequivalent thereto can be a protein to which various modifications areadded, such as the physiological modification of sugar chains, labelingwith fluorescent, radioactive or other substances, or fusion with otherproteins. In particular, in the recombinants described hereinafter,there is a possibility of differences in modifications arising due tothe sugar chains depending on the host in which it is expressed. Yeteven if they have differences in the modification of sugar chains forexample, as long as they exhibit properties similar to the Synoviolinprotein disclosed in this specification, any of them are still theSynoviolin according to the present invention or functionally equivalentproteins.

Synoviolin can be obtained not only from biological materials, but alsofrom recombinants wherein a gene that encodes it is incorporated into anappropriate expression system. Synoviolin can be obtained by geneticengineering techniques if the aforementioned polynucleotides that encodeSynoviolin is incorporated into an appropriate expression system andexpressed. An example of a host/vector system to which the presentinvention applies includes the expression vector pGEX-5X-3 and E. coli.pGEX-5X-3 can express a foreign gene as a fusion protein withglutathione S-transferase (GST) (Gene (1988) 67:31-40). Accordingly,when pGEX-5X-3 containing a gene that encodes Synoviolin is transformedinto an E. coli strain such as BL21 under heat shock and cultured for anappropriate length of time, and then isopropylthio-β-D-galactoside(IPTG) is added, the expression of GST-fusion Synoviolin is induced. Agene that encodes Synoviolin can be obtained by amplification by PCR orthe like with a cDNA library of synovial cells or the like as atemplate. Because the GST according to the present invention is adsorbedto Glutathione Sepharose 4B, the expression product can be easilyisolated and purified by affinity chromatography.

Other examples of host/vector systems used to obtain recombinants ofSynoviolin include the following. First, when a bacterium is to be asthe host, expression vectors of fusion proteins using a histidine tag,HA tag, Flag tag or the like are commercially available. As for yeast,yeast of the genus Pichia is known to be effective in the expression ofprotein with sugar chains. From point of the addition of sugar chains,expression systems that utilize a baculovirus vector with an insect cellas the host are also useful (Bio/Technology, (1988) 6:47-55). Moreover,transfection of vectors using CMV, RSV or SV40 or other promoters isperformed using cells of mammals, and these host/vector systems can eachbe used as an expression system for Synoviolin. In addition, genes canalso be introduced using retrovirus vectors, adenovirus vectors,adeno-associated virus vectors or other virus vectors.

The novel protein Synoviolin provided by the present invention, andimmunologically equivalent proteins are useful in the diagnosis of RA byutilizing its immunological characteristics. Antibodies that recognizeSynoviolin are detected with a high frequency in the blood of RApatients, and are substantially not detected in the blood of healthypersons. Accordingly, performing an immunological analysis of theantibodies of a subject using the Synoviolin according to the presentinvention as an antigen gives useful information for the diagnosis ofRA. To wit, if antibodies that react with Synoviolin are detected in thebody fluids of a subject, then the subject may be diagnosed to have RA.

Many methods of performing the immunological analysis of antibodies aregenerally used. The most popular method among the various methods ofperforming immunological analysis of antibodies is the method reactingan antigen sensitization plate with antibodies in the sample anddetecting, using an antibody-specific labeled antibody, the antibodiesthat are trapped on the surface of the plate and that is subjected todetection (Immunochemistry, (1971) 8:871-879). A method that uses anenzyme as a labeling is called the ELISA method and is in broad use. Inaddition, there is also a known method of mixing the sample with latexparticles to which antigens are adhered and detecting antibodies as animmunological agglutination reaction (Am. J. Med., (1956) 21:888-892).The immunological agglutination reaction is a method that permits rapidanalysis with a single reagent, and this is a preferable method forlarge-scale screening.

Moreover, immunochromatography has recently become widespread as asimple analytical method. In order to apply this method to a method ofimmunological analysis of antibodies, a reaction system is constructedwhere the reaction between labeled Synoviolin and anti-Synoviolinantibodies is blocked by the antibodies in the sample. Specifically, forexample, this is arranged so that the labeled Synoviolin and the samplecan first contact with each other and then this can contact with thereagent component of anti-Synoviolin antibodies by a chromatographicdevelopment. If Synoviolin antibodies are present in the sample, thenthe labeled Synoviolin has already reacted, and accordingly, it cannotreact any more with the anti-Synoviolin antibodies, which are thereagent component. By fixing the anti-Synoviolin antibodies andobserving the state of the reaction of labeled Synoviolin in the regionwhere the antibodies have been fixed, it is possible to perform animmunoassay by merely dripping samples.

In many immunoassays, it is possible to analyze antibodies according tothe class of the antibodies. If necessary, information regarding aspecific class of antibodies can be obtained by combining antibodiesthat can recognize classes of immunoglobulin such as IgG and IgM. Ininfectious diseases, a transition is observed where the IgM antibodymeasurements increase in the first stage of infection, and thereafter,the IgM antibody measurements decrease while the IgG antibodymeasurements increase. Such class-by-class antibody measurements may beassociated with clinical symptoms of RA in the present invention also.More specifically, class-by-class measurements of antibodies may belinked to the judgment of drug efficacy or the prediction of RA onset.

In the detection of antibodies, methods are often adopted that use notonly the antigen molecule itself but also chemically synthesizedoligopeptides as the antigen. This is because using an analysis systemthat is specific to a particularly superior epitope or an epitope thathas some clinical meaning is less affected by non-specific reactions.This approach is effective for Synoviolin also. Specifically, it ispossible to determine the domain that functions as an epitope, based onthe aforementioned method of obtaining the immunologically active domainpeptide. Epitopes are known to consist of at least three amino acidresidues in some cases. In addition, immunological distinction fromother proteins is said to be possible with at least 8 amino acidresidues. Accordingly, fragments that consist of at least 8 consecutiveamino acid residues, normally 9 amino acid residues, preferably 10 aminoacid residues, and more preferably 11 amino acid residues selected fromthe amino acid sequence of Synoviolin and that react with antibodies ina patient's blood serum are preferable as the antigen for detectingantibodies in the present invention. Moreover, methods of increasing theimmunological reactivity of epitope-forming oligopeptides by addingvarious modifications to the oligopeptides are also known to personsskilled in the art. For example, the modification of adding an inactiveprotein such as human blood serum albumin or a meaningless amino acidsequence contributes to improving the immunological reactivity.

The Synoviolin, which is useful in the method of detecting RA accordingto the present invention, functionally equivalent proteins thereto, orpartial peptides thereof can be used as immunological analysis reagentsfor analyzing antibodies that recognize these molecules. Theimmunological analysis reagents according to the present invention areuseful for the diagnosis of RA and the judgment of the effectiveness oftreatment.

The Synoviolin according to the present invention also makes possiblethe development of vaccines for the purpose of curing or preventing RA.Since Synoviolin is thought to induce the hyperplasia of synovial cellsby binding to its ligand, the treatment and prevention of RA can beachieved by providing a vaccine that gives an antibody that blocks thebinding of Synoviolin to its ligand. Typical methods of obtaining aSynoviolin vaccine are methods for formulating by combining mainly thedomain peptide serving as the epitope of Synoviolin, with an adjuvant ora carrier protein that gives an immune stimulus due to the domainpeptides of Synoviolin, which originally a human protein.

Moreover, the present invention provides antibodies that recognizeSynoviolin. Antibodies against Synoviolin can be obtained by knownmethods by taking as the immunogen the Synoviolin according to thepresent invention, its immunologically equivalent proteins or fragmentsthereof. Polyclonal antibodies may be obtained by ordinary immunemanipulation (Harlow, E. & Lane, D.; Antibodies: A Laboratory Manual,Cold Spring Harbor, N.Y., 1988), while monoclonal antibodies may beobtained by cloning antibody-producing cells (Kohler, G. & Milstein, C.,Nature (1975) 256:495-497). Monoclonal antibodies are important toolsfor achieving high sensitivity and specificity in immunoassays.

In immunization, an immune animal is immunized with the Synoviolinaccording to the present invention (or an immunologically equivalentprotein thereto or fragment thereof) along with an appropriate adjuvant.Synoviolin fragments that are useful as an immunogen include peptidescomprising the following amino acid sequences:

Syno-P3 (SLALTGAVVAHAYYC/SEQ ID NO: 3), Syno-P2 (TCRMDVLRASLPAQS/SEQ IDNO: 4), and Syno-P1 (GAATTTAAGTSATAC/SEQ ID NO: 5).

The immunogens prepared by linking these peptides to a carrier proteinare specific to Synoviolin and give antibodies that have adequatebinding affinity. Keyhole lympet hemocyanin (KLH), bovine serum albumin(BSA) or the like can be used as the carrier protein used to obtain theimmunogen. The immune animals typically used include a rabbit, mouse,rat, goat or sheep. The adjuvants typically used include Freund'scomplete adjuvant (FCA) and the like (Adv. Tubercl. Res., (1956)1:130-148). By adding immunity at appropriate intervals and drawingblood upon confirming the increase in the antibody titer, it is possibleto obtain antiserum. Moreover, by purifying its antibody fractions, itis possible to obtain purified antibodies.

Alternatively, monoclonal antibodies can be obtained by collectingantibody-producing cells and cloning them by cell fusion or othermethods. These antibody-producing cells include those derived fromimmune animals and also antibody-producing cells collected from RApatients that produce auto-antibodies against Synoviolin. Moreover, itis possible to construct chimeric antibodies or humanized antibodiesbased on the antibody genes of monoclonal antibody-producing cellsderived from immune animals thus obtained. When antibodies areadministered to humans, animal antibodies are not preferable becausethey will be eliminated as foreign matter. For this reason, chimericantibodies wherein human antibodies are substituted for the constantregions of strongly antigenic antibodies, or humanized antibodieswherein human genes are substituted for not only the constant regionsbut also the framework of the variable regions are required. At thispoint, by using the variable regions of antibodies derived fromantibody-producing cells of RA patients, it is possible to reconstructhuman-type antibodies, and accordingly, it is possible to constructhighly safe antibodies more easily.

The chimeric antibodies or humanized antibodies that recognizeSynoviolin provided based on the present invention are useful in a drugdelivery system (DDS) that targets the synovial cells of RA patients. Ina DDS that uses antibodies that recognize Synoviolin according to thepresent invention, Fas ligands or anti-SL antibodies or the like can beshown to be substances expected to be useful by linking to antibodies.

Alternatively, the antibodies of the present invention are useful in thedetection of Synoviolin. Synoviolin is overexpressed in the synovialtissue of RA patients. Accordingly, the detection of Synoviolin insynovial cells, synovial tissue or body fluids gives importantinformation for the diagnosis of RA. Specifically, when Synoviolin isdetected in synovial tissue or blood, RA is thought to be advanced. Theantibodies of the present invention can be used as reagents for theimmunological detection of Synoviolin. Methods of using antibodies toimmunologically detect the proteins present in tissue or blood areknown. Reagents for the immunological analysis that contain theantibodies according to the present invention are useful in thediagnosis of RA and the determination of the effectiveness of treatment.

In addition, the antibodies according to the present invention can beused for the separation or detection of cells that express Synoviolin.The Synoviolin protein according to the present invention is observed inAER in development, and is also expressed strongly in undifferentiatedmesenchymal cells that become the anlage of synovial membrane,cartilage, bone and limbs. Accordingly, Synoviolin can be used as amarker of AER and undifferentiated mesenchymal cells. To wit, it ispossible to detect and separate AER and undifferentiated mesenchymalcells using the expression of Synoviolin as an index. The antibodies areappropriately labeled by fluorescence or the like. For example,antibodies against Synoviolin can be used in cell sorting or the like toseparate cells that express Synoviolin. The separated undifferentiatedmesenchymal cells are useful in the in vitro formation of bone andcartilage, or the reconstruction of joints.

The stroma of bone, cartilage, muscle, tendons, fat, bone marrow and thelike are formed from undifferentiated mesenchymal cells in vitro or invivo (Kuznetsov, S. A., et al., J. Bone Miner. Res. (1997) 12:1335-1347;Prockop, D. J., Science (1997) 276:71-74; Thompson, C. M. and Young, R.A., Proc. Natl. Acad. Sci. USA (1995) 92:4587-4590; Caplan, A. I., J.Orthop. Res. (1991) 9:641-650; Friedenstein, A. J., Int. Rev. Cytol.(1976) 47:327-359; Owen, M. and Friedenstein, A. J., in “Cell andMolecular Biology of Vertebrate Hard Tissues,” D. Evered and S. Harnett,Eds., Wiley, Chichester, UK, (1988) pp. 42-60; Friedenstein, A. J., etal., Cell Tissue Kinet. (1987) 20:263-272; Ashton, B. A., et al., Clin.Orthop. Relat. Res. (1980) 151:294-307; Bab, I, et al., Clin. Orthop.Relat. Res. (1984) 187:243-254; Haynesworth, S. E., et al., Bone (1992)13:81-88; Caplan, A. I., Clin. Plast. Surg. (1994) 21:429-435; also see,for example, the http web site of Genzyme, www.genzymebiosurgery.com/).

For example, it is possible to differentiate undifferentiatedmesenchymal cells in vitro and form cells of adipocytic lineage,chondrocytic lineage and osteocytic lineage (Pittenger, M. F., et al,Science (1999) 284:143-147).

Differentiation to adipocytes can be induced by, for example, treatmentwith 1-methyl-3-isobutylxanthine, dexamethasone, insulin andindomethacin (Pittenger, M. F., U.S. Pat. No. 5,827,740, 1998).Differentiation to chondrocytes can be performed by, for example, usingcentrifugation or the like to make the cells into minute clumps and thenstimulating with transforming growth factor (TGF)-β3 in a culture mediumthat contains no blood serum (Mackay, A. M., et al., Tissue Eng. (1998)4:415-428; Yoo, J. U., et al., J. Bone Joint Surg. Am. (1998) 80A:1745-1757). Differentiation to osteocytes can be induced bydexamethasone, β-glycerophosphate and ascorbic acid in the presence of10% fetal calf serum, for example (Kuznetsov, S. A., et al., J. BoneMiner. Res. (1997) 12:1335-1347; Prockop, D. J., Science (1997)276:71-74; Thompson, C. M. and Young, R. A., Proc. Natl. Acad. Sci. USA(1995) 92:4587-4590; Caplan, A. I., J. Orthop. Res. (1991) 9:641-650;Friedenstein, A. J., Int. Rev. Cytol. (1976) 47:327-359; Owen, M. andFriedenstein, A. J., in “Cell and Molecular Biology of Vertebrate HardTissues,” D. Evered and S. Harnett, Eds., Wiley, Chichester, UK, (1988)pp. 42-60; Bruder, S. P., et al., J. Cell. Biochem. (1997) 64:278-294;Jaiswal, N., et al., J. Cell. Biochem. (1997) 64:295-312; Bruder, S. P.,et al., J. Bone Miner. Res. (1998) 13:655-663).

In addition, regarding the in vivo case also, for example,undifferentiated mesenchymal cells can be transplanted in utero anddifferentiated into cartilaginous cells, fat cells, muscle cells,cardiac muscle cells, bone marrow stromal cells and thymus stromal cells(Liechty, K. W., et al., Nature Medicine (2000) 6:1282-1286). By thesemethods, it is possible to reconstruct tissue in vitro or in vivo fromthe separated undifferentiated mesenchymal cells. The reconstructedtissue or organs is expected to have application in regenerativemedicine.

In addition, because Synoviolin is overexpressed in rheumatoid synovialcells, it can be used as a cell marker for rheumatoid synovial cells. Ifthe antibodies according to the present invention are used as reagentsfor the separation or detection of cells, then the antibodies can becombined with other solvents or solutes to form a composition. Forexample, it can be combined with distilled water, pH buffers, salts,proteins, surfactants and the like.

Synoviolin is overexpressed in the synovial tissue of RA patients. Inaddition, antibodies that recognize Synoviolin (auto-antibodies) aredetected with a high frequency in the blood of RA patients. On the otherhand, Synoviolin antibodies are substantially undetectable in the bloodof healthy persons. Moreover, Synoviolin suppresses the growth ofcultured synovial cells in vitro. This is thought to be becauseSynoviolin competes with ligands that promote synovial cell growth.Based on this information, the following mechanism can be expected. Towit, the overexpression of Synoviolin in synovial cells promotes thebinding of Synoviolin, which has a growth-promoting action on synovialcells, to ligands, and as a result, growth of synovial cells ispromoted. Moreover, the hyperplasia of these synovial cells itself isnothing other than the pathology of RA.

Based on the aforementioned knowledge, the present invention provides amethod of detecting or method of diagnosing rheumatoid arthritiscomprising the following steps of:

i) detecting markers for rheumatoid arthritis present in a biologicalspecimen from a subject, and

ii) associating the results of detection of step i) with rheumatoidarthritis.

The markers used in the method of detecting or method of diagnosingrheumatoid arthritis according to the present invention may be any ofthe following markers. The method of measuring these markers is asdescribed previously.

Synoviolin or a polynucleotide functionally equivalent to Synoviolin,

Synoviolin or a protein functionally equivalent to Synoviolin,

Synoviolin or a peptide functionally equivalent to Synoviolin,

Antibodies that bind to Synoviolin or a protein functionally equivalentto Synoviolin, and

Antibodies that bind to Synoviolin or a peptide functionally equivalentto Synoviolin.

For example, if antibodies that react with Synoviolin or a protein orpeptide functionally equivalent to Synoviolin are detected in a bloodsample taken from a patient, then the probability that the patient hasRA is high. Alternatively, the expression of Synoviolin or a proteinfunctionally equivalent to Synoviolin in synovial tissue taken from apatient indicates the hyperplasia of synovial tissue due to RA. Theexpression of a protein can be detected using the presence of theprotein or mRNA as an index.

In addition, the Synoviolin according to the present invention and thegene therefor provide new approaches to the development of drugs for thetreatment of RA, based on the above mechanism and such. First of all,with the Synoviolin according to the present invention, ligands ofSynoviolin can be detected using the binding activity to Synoviolin asan index. To wit, the present invention relates to a method of detectingthe binding activity to Synoviolin comprising the following steps of:

a) contacting a test compound with Synoviolin or a protein or peptidefunctionally equivalent to Synoviolin, and

b) observing the binding of the test compound to said protein orpeptide.

Moreover, it is possible to perform the screening of ligands forSynoviolin based upon the aforementioned detection method. The screeningmethod according to the present invention specifically comprises thefollowing steps of:

a) detecting the binding activity of test compounds to Synoviolin or aprotein functionally equivalent to Synoviolin by the aforementionedmethod of detecting the binding activity to Synoviolin, and

b) selecting the test compounds the binding activity of which is higherthan a control.

Candidate compounds for ligands include not only natural substances andvariants thereof but also low-molecular weight organic compounds.Binding between the aforementioned proteins and candidate compounds canbe detected directly by labeling the candidate compounds. Alternatively,this can be confirmed using the blocking of binding with a known SL asan index. To wit, the candidate compound is contacted with the proteinaccording to the present invention in the presence of molecules thatclearly exhibit the binding activity to proteins according to thepresent invention, such as S1-5. Alternatively, after the candidatecompound is contacted with the protein according to the presentinvention, it is possible, by further contacting with SL, to evaluatethe binding activity of the candidate compound. In the case where theblocking of binding is used as an index, only SL, which is alreadyknown, has to be labeled. Accordingly, this enables a simple screeningmethod.

As a control, it is preferable that the same manipulation as in step a)be performed in the absence of the test compound. Alternatively, thismay be a control where the test compound is present at a concentrationlower than that of step a). In addition, it is also possible to performthe same manipulation in step a) using a molecule that is known to bindto Synoviolin in place of the test compound, and thus select compoundsthat have a binding activity higher than that of that molecule.

In addition, screening methods for ligands based on the genes shown inExamples are also possible. For example, a commercial two-hybrid systemmay be used to screen a library containing genes that encode candidateligands for genes that encode proteins that bind to Synoviolin. Thismethod is an effective method of screening for natural ligands.Alternatively, it is possible to clone ligands by expression screeningusing a phage library into which cDNAs are incorporated and labeledSynoviolin. The present inventors used this screening method to discoverthe natural ligand of Synoviolin called SL. SL may possibly bind toSynoviolin on the surface of synovial cells and stimulate hyperplasia.Accordingly, the measurement of the level of SL in the blood maypossibly be associated with the pathology of RA. SL can be measuredbased on the binding activity to Synoviolin. Naturally, an immunoassaycan be performed with anti-SL antibodies, and SL can also be measured bya sandwich method that combines the two.

The present inventors confirmed that when Synoviolin is added tocultured synovial cells, it acts to suppress hyperplasia. This isexplained in terms of the neutralization of SL in the culture medium asfollows: the blocking of the binding of Synoviolin to its ligandconceivably leads to the suppression of hyperplasia of synovial cells,thereby conferring the effect of treating RA. Ligands that can beobtained by the screening method of the present invention competitivelyblock the binding of Synoviolin to its natural ligand, and accordingly,they can be expected to have the activity of effectively suppressing thehyperplasia of RA synovial cells (as an antagonist).

In addition, the Synoviolin ligands that can be obtained by thescreening method of the present invention can be expected to have theactivity of stimulating the activity of Synoviolin (as an agonist) inthe same manner as the aforementioned SL. Ligands that stimulateSynoviolin are useful as an agent that stimulates Synoviolin or an agentthat promotes bone formation. More specifically, ligands that stimulateSynoviolin can be used as drugs to treat osteoporosis, bone disruption,sports injuries or the like.

These methods of detecting binding activity and screening methods can beexpanded so that the present invention further provides a method ofdetecting the activity of blocking the binding of Synoviolin or afunctionally equivalent protein to the Synoviolin ligand, and a methodof screening compounds. The method of detecting the activity of blockingthe binding of Synoviolin to the Synoviolin ligand based on the presentinvention comprises the following steps of:

a) contacting the Synoviolin or a protein or peptide functionallyequivalent to Synoviolin with its ligand in the presence of a testcompound, and

b) detecting the ligand and/or test compound that binds to said proteinor peptide.

Moreover, based on the above-mentioned detection method, the presentinvention provides a method of screening for compounds that block thebinding of Synoviolin or a protein functionally equivalent thereto tothe Synoviolin ligand. To wit the present invention relates to thefollowing screening method comprising the steps of:

a) detecting the activity of test compounds to block the binding ofSynoviolin or a protein functionally equivalent thereto to its ligand bythe aforementioned detection method, and

b) selecting the test compounds said blocking activity of which ishigher than a control.

As a control, it is preferable that the same manipulation as in step a)be performed in the absence of the test compound. Alternatively, thismay be a control where the test compound is present at a concentrationlower than that of step a). In addition, for example, it is alsopossible to perform the same manipulation in step a) using a moleculethat is known to block the binding between Synoviolin and its ligand inplace of the test compound, and thus select compounds that have abinding activity higher than that of that molecule.

By this screening, it is possible to obtain compounds that act as anantagonist to Synoviolin or functionally equivalent proteins thereto.Examples of ligands of Synoviolin include the SL (S1-5) recited inExamples. Specifically, the S1-5 proteins identified by accession numberAAA65590, 138449, NP_(—)061489, NP_(—)004096 or Q12805, or similarproteins can be used as long as they have the activity of binding to theSynoviolin protein (Lecka-Czernik, B., et al., Mol. Cell. Biol. (1995)15:120-128; Heon, E., et al., Arch. Ophthalmol. (1996) 114:193-198;Ikegawa, S., et al., Genomics (1996) 35:590-592; Katsanis, N., et al.,Hum. Genet. (2000) 106:66-72; Giltay, R., et al., Matrix Biol. (1999)18:469-480; Stone, E. M., et al., Nat. Genet. (1999) 22:199-202). Thecontact between Synoviolin and Synoviolin ligand can be made before,after or at the same time that the candidate compound is applied.

The compounds to be screened here are those that are thought to bind toSynoviolin and block the binding of Synoviolin to the ligand, and thosethat block the ligand. Compounds that bind to Synoviolin can be screenedby labeling the ligand and by making it compete with the candidatecompound. If a compound that binds to the ligand is a candidate, thenthe opposite is performed. In each screening, it is preferable thatradioactive isotopes are used for labeling, because their effect onactivity is small. An antagonist of Synoviolin thus obtained is presumedto have the action of suppressing the hyperplasia of synovial cells, andcan be expected to have the effect of treating RA.

In addition, the Synoviolin ligand S1-5 is suggested to be a causal genefor Malattia Leventinese (ML) and Doyne honeycomb retinal dystrophy(DHRD) (Stone, E. M., et al., Nature Genetics (1999) 22:199-202). Thesediseases have symptoms similar to age-related macular degeneration (AMD)wherein deposits known as drusen occur. Because of these, there is apossibility that Synoviolin contributes to ML and DHRD. Diagnosing MLand DHRD can be performed by investigating the mutations andpolymorphism of synoviolin. In addition, compounds that act as ligandsof Synoviolin obtainable by the screening according to the presentinvention, compounds that block the interaction between Synoviolin andS1-5 and the like are expected to have use as drugs that contribute tothe prevention or treatment of these diseases.

In addition, it is possible to use the Synoviolin according to thepresent invention to evaluate the activity of a compound to regulatesignal transduction via Synoviolin, or to screen for compounds thatregulate signal transduction via Synoviolin. Specifically, the presentinvention provides a method of detecting the activity of a test compoundto regulate signal transduction via Synoviolin, comprising the followingsteps of:

a) contacting the test compound with Synoviolin either in the presenceof, or in the absence of, the Synoviolin ligand, and

b) detecting signal transduction via Synoviolin.

In addition, the present invention relates to a method of screening forcompounds that have the activity of regulating signal transduction viaSynoviolin, comprising the following steps of:

a) detecting the activity of test compounds to regulate signaltransduction via Synoviolin by the aforementioned method, and

b) selecting the test compounds said regulation activity of which ishigher than a control.

As a control, it is preferable that the same manipulation as in step a)be performed in the absence of the test compound. Alternatively, thismay be a control where the test compound is present at a concentrationlower than that of step a). In addition, for example, it is alsopossible to perform the same manipulation in step a) using a moleculethat is known to have the activity of promoting or blocking the signaltransduction via Synoviolin in place of the test compound, and thusselect compounds that have a regulating activity higher than that ofthat molecule.

In the present invention, signal transduction via Synoviolin is definedto be that the stimulus applied to Synoviolin is transduced to differentmolecules. There is no limitation on the type of stimulus. Many modes ofsignal transduction are known to be present in the body. Arepresentative example of signal transduction is the regulation ofactivity by the modification of proteins. For example, the activity ofcertain types of proteins is regulated by phosphorylation oracetylation. In addition, the activity of a protein is known to becontrolled by its cleavage. In order for a protein to be cleaved in amore specific manner, the presence of ubiquitin or other molecules isimportant. Signal transduction can be detected by using as an index thechange in the activity or structure of molecules that constitute signaltransduction where the change is generated by the transduction ofsignals. Alternatively, signal transduction can be detected using as anindex the formation of complexes for signal transduction.

Examples of signal transduction especially include phosphorylation ordephosphorylation signals. Many of cell proliferation signals are knownto be transduced to downstream signal molecules via protein modificationbased on protein phosphorylation or dephosphorylation. Since theSynoviolin according to the present invention also has a cellproliferation action, this suggests that signal transduction viaSynoviolin is also transduced by phosphorylation of protein. In fact,the present inventors discovered the phosphorylation action ofSynoviolin expression. Accordingly, it is possible to measure signaltransduction via Synoviolin by detecting the phosphorylation of protein.

Receptors involved in cell proliferation or differentiation have thefollowing domains as enzyme active sites (Jikken IgakuBessatsu•Bioscience Yōgo Raiburari, Kaiteiban Saitokain Zōshoku Inshi[Experimental Medicine, Supplement—Bioscience Terminology Library,Revised Version: Cytokines/Growth Factors], Yodosha Co., Ltd., 1998):

the tyrosine kinase domain (VEGF receptor, PDGF receptor, HGF receptor,EGF receptor, etc.),

the tyrosine phosphatase domain (RPTP, etc.), and

the serine/threonine kinase domain (TGFβ receptor, etc.).

Synoviolin is predicted to keep these enzyme activities either directlyor indirectly. The phrase “having an enzyme activity indirectly” refersthat there is no enzyme activity site in the Synoviolin molecule, but amolecule that is associated with Synoviolin has an enzyme activity.Known examples of such molecules include the TNF receptor, GM-CSFreceptor and the like. Accordingly, by detecting the phosphorylationactivity on tyrosine, serine and/or threonine, for example, it ispossible to evaluate signal transduction via Synoviolin. At this time,by evaluating the action of the test compound in the presence ofSynoviolin ligand, it is possible to evaluate the effect of the testcompound on the signal transduction of Synoviolin triggered by aSynoviolin ligand. Specifically, it is possible to detect the activityof blocking or suppressing the signal transduction via a Synoviolinligand to Synoviolin. As the Synoviolin ligand, the Synoviolin ligandS1-5 described herein can be used. Alternatively, by evaluating theaction of the test compound in the absence of Synoviolin ligand, it ispossible to evaluate the stimulation activity of the test compound toSynoviolin.

In order to detect the phosphorylation of protein, for example,Synoviolin-expressing cells together with the test compound and [³²P]orthophosphate are incubated both in the presence of, and in the absenceof the Synoviolin ligand. Next, by immune precipitation, thephosphorylated protein is recovered from this cytolysis product. Afterfractionation by SDS-PAGE, the phosphorylation of the protein thusrecovered can be detected by autoradiography. Phosphorylated amino acidscan be identified by TLC or other known methods of peptide analysis.

Alternatively, phosphorylated tyrosine antibodies or other antibodiesspecific to phosphorylated protein can be used to detect thephosphorylation of specific amino acids.

Typically, the phosphorylation of signal-transduction factors in thecell is transduced sequentially to a plurality of molecules. To wit, aseries of transduction paths constitutes a cascade. For this reason, byevaluating changes in the phosphorylation level of the entire protein inthe cell, it is possible to compare the magnitude of the phosphorylationsignal occurring in the cell. Methods of evaluating the phosphorylationlevel of the total protein in the cell are known. For example, aftercells are stimulated with Synoviolin ligand or the like, the protein isfractionated by SDS-PAGE and blotted on a filter, and then thephosphorylation level of the entire protein can be evaluated by Westernblot using anti-phosphorylated tyrosine antibodies or the like. Inaddition, for example, the cells are labeled with [³²P] orthophosphate,and the cells are stimulated with Synoviolin ligand or the like. Then,the cell protein is expanded with two-dimensional electrophoresis. Theprotein is stained with Coomassie blue and autoradiography is performed.By detecting the phosphorylated spot, it is possible to evaluate thephosphorylation level.

Alternatively, it is possible to specifically measure the change in thephosphorylation level in the phosphorylated protein that is thesubstrate of Synoviolin. The phosphorylated protein that is thesubstrate of Synoviolin is, for example, recovered from thephosphorylated spot in the aforementioned two-dimensionalelectrophoresis and can be identified by microsequencing or massspectrometry. Changes in the phosphorylation level of the identifiedsubstrate protein is subjected to, for example, immune precipitationusing antibodies specific to the substrate protein, and afterfractionation by SDS-PAGE, the intake of [³²P] may be measured byautoradiography or evaluated by Western blotting usinganti-phosphorylated tyrosine antibodies (Baio Manyuaru Shirīzu—BunshiSeibutsugaku Kenkyū no Tame no Tampaku Jikken Hō [Bio ManualSeries—Protein Experimental Methods for Molecular Biology Research],Tadaomi Takenawa, Masaki Inagaki eds.).

Examples of cells used in the aforementioned method include synovialcells (e.g. RTF) and cells into which the synoviolin gene is exogenouslytransferred. If the level of phosphorylation or dephosphorylation due toSynoviolin decreases for a certain test compound, then the compound isjudged to be a compound that blocks signal transduction via Synoviolin.In addition, if the level of phosphorylation or dephosphorylation due toSynoviolin increases for a certain test compound, then the compound isjudged to be a compound that promotes signal transduction viaSynoviolin.

For example, in the case where Synoviolin functions as a receptor-typetyrosine kinase and the downstream molecule is activated via tyrosinephosphorylation to transduce a signal, if tyrosine phosphorylation issuppressed by the test compound, then this compound is judged to be acompound that blocks signal transduction via Synoviolin. In addition,the present invention relates to a method of blocking signaltransduction via Synoviolin using, for example, tyrosine kinase,tyrosine phosphatase or serine/threonine kinase or other protein kinaseor phosphatase blocker.

In addition, another good example of signal transduction via Synoviolinis ubiquitination signals. A protein structure prediction system (SMART:Simple Modular Architecture Research Tool (also see the http web sitesmart.embl-heidelberg.de/) Schultz et al., Proc. Natl. Acad. Sci. USA(1998) 95:5857-5864; Schultz, et al., Nucleic Acids Res. (2000)28:231-234) demonstrated the presence of a ring finger motif inSynoviolin (Joazeiro, C. A., et al., Science (1999) 286:309-312). Thismotif is known to be present in the E3 ubiquitin-protein ligase relatedto the decomposition of protein. In addition, the ring finger motif isthought to be the binding site for E2 ubiquitin-conjugating enzymes.

Accordingly, by detecting the ubiquitination signal due to Synoviolin,it is possible to evaluate signal transduction via Synoviolin. Theubiquitination signal is evaluated, for example, by detectingubiquitination of the substrate protein using anti-ubiquitin antibodies.In addition, the binding of Synoviolin to E2 ubiquitin-conjugatingenzyme or substrate protein, or the Synoviolin-containing ubiquitinligase complex may also be detected. Specifically, for example, cellstransfected with a vector that expresses tagged Synoviolin are rupturedand [³²P]-labeled ubiquitin is added. After reaction is allowed toproceed, immunoprecipitation is performed with anti-tag antibodies. Theubiquitin ligase activity of Synoviolin can be detected by SDS-PAGE andperforming autoradiography (Hashizume, R., et al., J. Biol. Chem. (2001)276:14537-14540).

Changes in the level of ubiquitination in the substrate protein ofSynoviolin can be measured specifically. The substrate protein ofSynoviolin can be identified by, for example, yeast two-hybrid screeningusing Synoviolin as a bait. Changes in the ubiquitination level of theidentified substrate protein can be evaluated as follows: the taggedsubstrate is purified, and purified E1, E2, E3 and ubiquitin are addedthereto. After reaction is allowed to proceed, immune precipitation isperformed with anti-tag antibodies, and staining is performed withanti-ubiquitin antibodies (Yokouchi, M., et al., J. Biol. Chem. (1999)274:31707-31712).

If the activation of the ubiquitination signal by Synoviolin decreasesfor a certain test compound, then the compound is judged to be acompound that blocks signal transduction via Synoviolin. In addition, ifthe activation of the ubiquitination signal increases for a certain testcompound, then the compound is judged to be a compound that promotessignal transduction via Synoviolin. For example, compounds that inhibitthe interaction between Synoviolin and E2 ubiquitin activation enzymecan effectively inhibit the ubiquitination signal via Synoviolin. Inaddition, the present invention provides a method of using a inhibitorof enzymes related to the ubiquitination signal to shut off signaltransduction via Synoviolin. For example, by applying the E2 ubiquitinconjugating enzyme or the E3 ubiquitin ligase inhibitor to cells, it ispossible to block signal transduction via Synoviolin.

The method described above can be used to select compounds that have theactivity of regulating signal transduction via Synoviolin. Compoundsthat block signal transduction via Synoviolin are useful as agents forthe treatment of diseases caused by the activation of Synoviolin. Forexample, compounds that block signal transduction via Synoviolin areuseful in synovial hyperplasia blocking. By administering thesecompounds, it is possible to suppress synovial hyperplasia and thus itis possible to prevent or treat diseases such as RA that involvesynovial hyperplasia. In addition, these compounds may also be used asdrugs for treating ML and DHRD. Alternatively, compounds that promotesignal transduction can be used as an agent that stimulates Synoviolin,or agent that promotes bone formation, etc. For example, they can beused as drugs for treating osteoporosis, bone disruption, sportsinjuries or the like.

Based on the discovery of the synoviolin gene, the following newresearch becomes possible regarding RA and other diseases in whichSynoviolin is involved. First, it is possible to determine the structureof the promoters or enhancers that control the expression of Synoviolin.To wit, it is possible to advance the cloning of the genome based on thenucleotide sequence of the synoviolin gene shown in SEQ ID NO: 1, andanalyze the sequence of the expression control domain. The thus-obtainedtranscription regulation domain for synoviolin can be used in the searchfor a transcription regulating factor for synoviolin.

In addition, in the synoviolin knock-out animal according to the presentinvention, if a marker gene is knocked in and the marker gene isexpressed under the control of the endogenous promoter of the synoviolingene, it is possible to perform a screening for drugs that control theexpression of the synoviolin gene, using this animal or theanimal-derived cells with the expression of the marker gene as an index.For example, if the recognition sequence for the transcriptionregulating factor is given as a double strand, then it functions as adecoy nucleic acid drug.

In addition, the polynucleotide according to the present invention maybe used to examine, in animals, the biological role of the proteinaccording to the present invention. In order to do this, for example,the DNA according to the present invention is introduced and the proteinaccording to the present invention is overexpressed or expressed atdifferent locations (or expressed at different times). Thus, its rolecan be examined by verifying its effect. A gene can be transferred intothe entire body by preparing a transgenic animal of the DNA according tothe present invention. Alternatively, through gene targeting and theadministration of antisense oligonucleotides, ribozymes and the like,loss-of-function experiments on the suppression of the expression andfunctions of the DNA of the present invention are also effective. Towit, the present invention provides transgenic non-human vertebrate inwhich the expression of the DNA of the present invention is modified orsaid modifications can be induced. Expression may be modified incomparison to that of the wild type or, in the case where modificationis induced, expression may be modified in comparison to that before theinduction.

The transgenic animals according to the present invention includeanimals wherein exogenic nucleic acids are transferred into the genome.In addition, the “expression of DNA” may be at the DNA transcriptionlevel or at the translation level of transcripts. In addition, the“induction of modifications” may include the induction of modificationby external stimulus or modification of stage-specific expression, orthat expression is modified in later generations due to cross-breeding.In addition, it includes the modification of expression in some cells ortissues. Examples of the transgenic non-human vertebrates according tothe present invention preferably include mammals (e.g., mouse, rat,hamster, rabbit, pig, goat, sheep, horse and bovine), while rodents,e.g. mouse and rat and the like are particularly used.

The transgenic non-human vertebrates according to the present inventioninclude transgenic non-human vertebrates into which DNA that encodes theprotein according to the present invention is exogenously introduced.Such transgenic animals can be produced by, for example, injecting, intoa fertilized egg, a vector that expresses the DNA that encodes theprotein according to the present invention.

The transfer of vectors can be performed by treatment with calciumphosphate after the mixing of vector and egg, electroporation, ormicroinjection under an inverted microscope, etc. In addition, thetransfection is also performed by transferring a vector according to thepresent invention into an embryonic stem cell (ES cell), and byperforming the microinjection of a selected ES cell into a fertilizedegg (blastocyst).

The fertilized egg thus obtained may be implanted into the fallopiantubes of a recipient in which a false pregnancy is induced throughmating with a vasectomized male individual, thereby obtaining a newborn.DNA is prepared from the tail of the newborn or the like and PCR is usedto confirm that the transferred DNA was kept (Brigid Hogan, et al.,eds., “Manipulating the Mouse Embryo: A Laboratory Manual,” Cold SpringHarbor Laboratory, 1994, Gordon, J. W., et al., Proc. Natl. Acad. Sci.USA (1980) 77:7380-7384; Jaenisch, R. and Mintz, B., Proc. Natl. Acad.Sci. USA (1974) 71:1250-1254). A heterozygote can be obtained from achimeric animal into which the genes are transferred into the germ lineby breeding with a normal animal. A homozygote can be obtained by thebreeding of two heterozygotes. The transgenic non-human vertebratesaccording to the present invention include these and their descendents.

Examples of the promoters used for expressing the DNA of the presentinvention in vivo include whole body expression type promoters andtissue-specific and stage-specific promoters.

Examples of whole body expression type promoters include β-actinpromoters and the like. For example, chicken β-actin promoter linked toa human cytomegalovirus enhancer contained in pCAGGS or the like may beused. In the case of preparing a transgenic animal wherein the DNAaccording to the present invention is expressed in a site-specific orstage-specific manner, a Cre-loxP system or the like can be used. Forexample, a transgenic animal having a Cre recombinase gene downstream ofa site-specific or stage-specific promoter is prepared and separately atransgenic animal having a vector in which DNA that encodes thepolypeptide of the present invention is linked downstream of ageneral-use promoter is prepared. At this time, a transcriptiontermination signal or the like or a stop codon sandwiched between a loxPpair is inserted between the promoter and the DNA that encodes thepolypeptide of the present invention. By mating two individuals, it ispossible to express the polypeptide of the present invention with theexpression of Cre.

In addition, the transgenic non-human vertebrates according to thepresent invention encompass transgenic non-human vertebrates wherein theexpression of DNA that encodes the endogenous protein of the presentinvention is suppressed. Such transgenic animals can be prepared by genetargeting, for example. In order to produce such transgenic non-humanvertebrates, for example, a targeting vector wherein some or all of theDNA according to the present invention is made defective bysubstitution, deletion, addition and/or insertion or the like isinserted into an embryonic stem (ES) cell, and cells wherein homologousrecombination with chromosome DNA has occurred are selected. Knownmethods of positive and negative selection can be performed in order toselect homologous recombinants. Examples of markers used for positiveselection include the neomycin resistance gene or other drug resistancegenes, while examples of markers used for negative selection include thediphtheria toxin (DT)-A gene, HSV-tk gene and the like. Southernblotting, PCR or the like can be used to select correctly recombinedcells. The cells thus obtained are inserted into a fertilized ovum atroughly the eight-cell stage or into the blastocoel of a blastocyst orthe like, and transferred to the uterus of a pseudopregnant femaleindividual prepared by mating with a vasectomized male. Genomic DNAanalysis of the newborn is performed in the same manner as above, and aheterozygote or homozygote can be obtained. Not only the target gene canbe knocked out, but another gene can also be knocked in. There is noparticular limitation on the knocked-in genes. Examples include the lacZgene or other marker genes.

In addition, transgenic non-human vertebrates wherein the expression ofthe DNA that encodes the endogenous protein according to the presentinvention is suppressed may be prepared using the antisense method orribozyme method. In the antisense method, a vector containing DNA thatencodes RNA complementary to the DNA transcription product that encodesthe protein according the present invention, or in the ribozyme method,for example, a vector containing DNA that encodes RNA that cuts thetranscription product of DNA that encodes the protein according thepresent invention is inserted into a embryonic stem cell of a mammal inthe same manner as above. This is injected into a mammal embryo andindividuals are obtained from the embryo.

Since Synoviolin induces the synovial hyperplasia symptoms of RA, thefollowing applications are conceivable for transgenic animals. To wit,after incorporating the synoviolin gene or SL gene into an appropriateanimal to form a transgenic animal, it can be used as a model for RA byinducing overexpression. In this transgenic animal, it is possible toproceed with the screening of drugs that control the mechanism ofsynovial hyperplasia. Alternatively, in animals wherein the RA symptomsdo not occur with human Synoviolin/SL, they can be utilized as a sourceof supply of Synoviolin or SL by inducing the overexpression of thesegenes.

Transgenic animals that express the synoviolin gene exhibit symptomscommon to RA such as arthritis accompanying synovial hyperplasia. Towit, these animals become rheumatoid arthritis model animals. Theseanimals can be used to perform the testing or screening of variouscompounds including candidate compounds for RA drugs. By administeringtest compounds to the transgenic animals, it is possible to observe theremission or exacerbation of symptoms to verify the effectiveness ofcompounds or perform screening. Examples of methods of using thetransgenic animals according to the present invention to perform testingor screening include the following methods.

A method of testing or screening for compounds that cause the remissionor exacerbation of joint abnormalities, which is a method comprising thesteps of: (a) administering a test compound to a transgenic non-humanvertebrate into which the DNA according to the present invention isexogenously inserted, and (b) evaluating the joint abnormalities of theanimal to which it was administered.

In addition, synoviolin gene knock-out animals can be used to examinethe side effects caused by the suppression of the action of Synoviolin,and can be used in the assay or screening of drugs that reduce theseside effects. In addition, by expressing Synoviolin locally ortransiently in knock-out animals, it is possible to perform the specificverification of the effects of Synoviolin. In addition, from theassociation of SL (S1-5) and ML/DHRD, it is possible that Synoviolin maycontribute to the intracellular signal transduction of SL, and thus,synoviolin knock-out animals may become models of ML or DHRD. Forexample, a tissue-specific or stage-specific (homozygous orheterozygous) knock-out of the synoviolin gene is conceivable.

A knock-in animal wherein a marker gene or the like is introduced at thetime of the knock-out of the synoviolin gene can be used to detect theactivity of the compound to increase or decrease the expression of thesynoviolin gene. To wit, the present invention relates to a method ofdetecting the activity of a test compound to regulate the expression ofthe synoviolin gene, comprising the following steps of:

a) applying the test compound to the aforementioned knock-in animal orknock-in cells, and

b) measuring the expression level of the marker gene.

This detection method can be used in the screening of compounds thatregulate the expression of the synoviolin gene. This method is ascreening method for compounds that regulate the expression of thesynoviolin gene, comprising the following steps of: a) applying the testcompound to the aforementioned knock-in animal or knock-in cells, b)measuring the expression level of the marker gene, and c) selectingcompounds that increase or decrease the expression of the knocked-ingene.

To wit, in the animal or cell to which the test compound is applied, theexpression of the marker gene is detected and compounds that increase ordecrease the expression of the marker gene are selected. The detectionof the expression of the marker gene in the case that LacZ is used as amarker can be performed by the method recited in Examples. By thismethod, in addition to the testing or screening using individuals, forexample, it is possible to use isolated organs or tissues and performsimilar testing or screening using cells obtained from transgenicanimals.

In screening using individuals, the test compound is administered via anappropriate route. The test compound may be administered by knownmethods of administration such as intravenous injection, hypodermicinjection, intramuscular injection, intraabdominal injection, oraladministration, rectal administration, nasal administration or the like.In the event that screening is performed using a test tube culturesystem, the test compound may be added to the culture medium, forexample. Alternatively, it may be injected into the cell bymicroinjection or other methods. In the event that the test compound isa gene, the naked DNA may be combined with a desired transfectionreagent or incorporated into a known expression vector and the gene isintroduced into the cell. Nucleic acids that include the sequence of thepromoter domain of the synoviolin gene are expected to act as a decoyand suppress the expression of Synoviolin.

The activity of regulating the expression of the synoviolin gene can bedetected, for example, by the following steps of:

a) contacting a test compound with an expression system that expresses areporter gene under the control of the endogenous promoter of Synoviolinor a polynucleotide functionally equivalent to Synoviolin, and

b) measuring the expression level of the reporter gene.

Moreover, based on this detection method, the screening of compoundsthat regulate the expression of the synoviolin gene can be performed. Towit, the present invention relates to a method of screening forcompounds that regulate the activity of endogenous promoters ofSynoviolin or polynucleotides functionally equivalent to Synoviolin,comprising the following steps of:

a) measuring the activity of test compounds to regulate the activity ofthe endogenous promoter of Synoviolin or a polynucleotide functionallyequivalent to Synoviolin by the aforementioned method of detectingactivity, and

b) selecting the test compounds that have a difference in said activityin comparison to a control.

As a control, the same manipulation as in step a) may be performed inthe absence of the test compound. Alternatively, this may be a controlwherein the test compound is present at a concentration lower than thatof step a). In addition, it is also possible to perform the samemanipulation in step a) using a different compound, for example, andthus select compounds that have an action higher than that of thatcompound. The expression of genes includes expression at thetranscription level or expression at the translation level. The genelinked downstream of the endogenous promoter of the synoviolin gene maybe the natural synoviolin gene itself or an artificially linked reportergene. The endogenous promoter activity of the synoviolin gene can bedetermined by detecting the transcription products or translationproducts of said gene by, for example, Northern hybridization using cDNAfragments of a gene linked downstream as the probe, RT-PCR, Westernblotting using antibodies to the proteins encoded by said gene, immuneprecipitation, ELISA or other methods.

In addition, by producing a construct wherein a reporter gene is linkeddownstream of the promoter of the synoviolin gene, and using thetransformed cell obtained by transfecting this into a cell, it ispossible to perform screening using the expression of the reporter geneas an index. Such a construct can be prepared by linking the desiredreporter gene downstream of the genome DNA in the upstream domain of thesynoviolin gene that contains the promoter of the synoviolin gene. Thereare no particular limitations on the reporter gene, and examples includeLacZ, chloramphenicol acetyl transferase (CAT), luciferase, GFP (greenfluorescent protein) and others. Compounds that decrease the expressionof the synoviolin gene are candidates for drugs for treating RA.

There are no particular limitations on the test compounds used in thetesting or screening according to the present invention, and examplesthereof include organic compounds, inorganic compounds, peptides,proteins, natural or synthetic low-molecular weight compounds, naturalor synthetic polymers, extracts of tissues or cells, microbial culturesupernatants and natural ingredients derived from plants or marineorganisms, but they are not limited to these. Expression products ofgene libraries or expression cDNA libraries and the like may also beused. In addition, compounds obtained by the aforementioned screening ofcompounds that bind to Synoviolin, or by screening of compounds thatblock the binding of Synoviolin to SL can also be administered as testcompounds.

There are no particular limitations on the method of administeringcompounds, and this can be performed in vitro by contact with cells,including addition to culture medium, or by the introduction into cellsusing a microinjector or transfection reagent, etc. This can beperformed in vivo by intraarterial injection, intravenous injection,hypodermic injection, intraabdominal administration, oraladministration, rectal administration, intramuscular administration, eyedrops, nasal administration, local injection into joints, etc., or othermethods known to persons skilled in the art. The compounds areadministered as an appropriate composition obtained by mixing withwater, physiological saline solution, buffer solution, salt, stabilizer,preservative, suspension agent or the like.

In addition, the screening of compounds that regulate the expression ofthe synoviolin gene can be performed using not only transgenic animalsbut also normal animals or cells or the like derived from those animals.For example, the present invention relates to a method of detecting theactivity of regulating the expression of Synoviolin or polynucleotidesfunctionally equivalent to Synoviolin, comprising the following stepsof:

a) culturing cells that express Synoviolin or a polynucleotidefunctionally equivalent to Synoviolin in the presence of a testcompound, and

b) measuring the expression level of said polynucleotide.

Moreover, based on this detection method, the screening of compoundsthat regulate the expression of the synoviolin gene can be performed. Towit, the present invention relates to a method of screening forcompounds that regulate the expression of Synoviolin or polynucleotidesfunctionally equivalent to Synoviolin, comprising the following stepsof:

a) detecting the activity of test compounds to regulate the expressionof Synoviolin or a polynucleotide functionally equivalent to Synoviolinbased on the aforementioned method of detecting activity, and

b) selecting the test compounds that have a difference in said activityin comparison to a control.

The expression level of Synoviolin or a polynucleotide functionallyequivalent to Synoviolin can be measured by the aforementioned method.In addition, all compounds that can be used as test compounds in theaforementioned and other screening methods can be used as the testcompound in this screening method. As a control, the same manipulationas in step a) can be performed in the absence of the test compound, asdescribed above.

Compounds identified by the testing or screening methods according tothe present invention become candidates for drugs for RA and otherdiseases in which Synoviolin is involved, and thus, they can be used forthe prevention or treatment of RA and other diseases. These compoundscan consist of the active ingredient appropriately combined with othersolutes or solvents to form pharmaceutical compositions. In the case ofusing, as a pharmaceutical agent, a compound isolated by the screeningmethod according to the present invention, it is possible to administerthe isolated compound itself directly to patients, or the compound maybe administered as a pharmaceutical composition prepared by knownpharmaceutical methods.

For example, it can be prepared and administered appropriately incombination with any pharmaceutically acceptable carrier or medium,specifically sterilized water, physiological saline solution, plantoils, emulsifiers, suspension agents or the like. The pharmaceuticalcomposition according to the present invention may take the form of anaqueous solution, tablet, capsule, troche, buccal tablet, elixir,suspension, syrup, nose drops, inhalation solution or the like. Thecontent of the compound may be determined appropriately. Administrationto patients may be performed typically by intraarterial injection,intravenous injection, hypodermic injection, oral administration,injection in the joint, etc., or other methods known to persons skilledin the art.

While the dosage varies depending on the weight and age of the patient,the method of administration, symptoms and the like, a person skilled inthe art would be able to select the dosage appropriately. The typicaldosage would differ depending on the effective blood concentration andthe metabolism time of the drug, but the daily maintenance dose isthought to be about 0.1 mg/kg to about 1.0 g/kg, or preferably about 0.1mg/kg to about 10 mg/kg, or even more preferably about 0.1 mg/kg toabout 1.0 mg/kg. Administration can be performed either at one time ordivided into several times. In addition, as long as said compound can beencoded by the polynucleotide, gene therapy can be performed byincorporating said polynucleotide into a gene therapy vector.

All prior art references cited herein are incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 indicates photographs of positive colonies in immunoscreening byanti-synovial cell anti-serum.

FIG. 2 indicates a photograph showing the expression of Synoviolinrecombinant protein in E. coli.

FIG. 3 indicates a photograph of an autoradiograph showing theSynoviolin protein expression translated from synoviolin cDNA in vitro.

FIG. 4 indicates a photograph of an autoradiograph showing the resultsof analysis of synoviolin gene expression by Northern blotting using thecDNA of Synoviolin as a probe.

FIG. 5 indicates photographs showing the results of Western blottingusing anti-synovial cell anti-serum on various cell extracts, and theresults of antibody absorption experiments with GST-partial Synoviolin.The arrow shows the absorbed band. The molecular weights of the variousbands are approximately 220, 185 and 140 kDa in order from the top.

FIG. 6 indicates a photograph of an autoradiograph showing the resultsof Western blotting using anti-synovial cell anti-serum on synovial cellextracts. The left lane (pre-immune) is rabbit anti-serum prior toimmunization of synovial cell, while the right lane (post-immune) issynovial cell anti-serum.

FIG. 7 indicates fluorescent microphotographs showing the results offluorescent immunostaining on synovial cell with anti-synovial cellanti-serum (A) and purified anti-synovial cell antibodies (B).

FIG. 8 indicates microphotographs showing the results of immunostainingusing anti-synovial cell anti-serum on synovial tissue, and the resultsof antibody absorption experiments with GST-partial Synoviolin.

FIG. 9 indicates microphotographs showing the results of immunostainingusing purified anti-synovial cell antibody on synovial tissue. Theresults using anti-serum purified by a GST affinity column (upper panel)and anti-serum purified by a GST-partial Synoviolin affinity column(lower panel) are shown.

FIG. 10 indicates a photograph of an autoradiogram showing the resultsof detection of anti-Synoviolin antibodies in various types of humanblood serum by Western blotting.

FIG. 11 indicates a photograph of an autoradiograph showing the resultsof analysis of the expression of the SL gene in synovial cells byNorthern blotting using the cDNA of SL as a probe.

FIG. 12 indicates a photograph of an autoradiograph showing the bindingbetween [³⁵S]-labeled HA-Synoviolin-HAHA and GST-SL fusion protein.

FIG. 13 indicates a diagram showing the results of analyzing the effectof Synoviolin on hyperplasia of synovial cells by MTT assay. GST-partialSynoviolin was used.

FIG. 14 indicates a diagram showing the structure of the synoviolin geneintroduction vector. Synoviolin is systemically expressed using aβ-actin promoter which has a CMV enhancer. The anti-Flag-tag antibodycan be used to confirm the expression of the Flag tag-fusion Synoviolinprotein.

FIG. 15 indicates photographs showing toe joints that exhibit arthritisin a transgenic mouse with the synoviolin gene. The appearance and softX-ray image of the toe of a Synoviolin forced-expression mouse areshown. A soft X-ray image of the toe of a normal mouse is shown at rightfor comparison. The Synoviolin forced-expression mouse exhibited markedswelling of the toe.

FIG. 16 indicates photographs showing the histological findings on toejoint that exhibits arthritis in a transgenic mouse with the synoviolingene. In the joint portions of toes that exhibited marked swelling,marked bone disruption and abnormal bone formation accompanying synovialhyperplasia were found.

FIG. 17 indicates photographs showing the histological findings onnormal toe joint of a gene-introduced mouse. No abnormal jointcartilage, bone disruption or synovial hyperplasia were found. The lowerright panel shows the results of immunostaining with anti-Flagantibodies. No positive signal was observed.

FIG. 18 indicates photographs that show the expression of Synoviolin intoe joints that exhibit arthritis in a synoviolin gene transgenic mouse.Immunostaining with anti-Flag antibodies was performed. The expressionof Synoviolin was found in the hyperplastic synovial tissue andcartilaginous cells formed in the joint areas of toes that exhibitedmarked swelling.

FIG. 19 indicates diagrams showing the structure of a targeting vectorto make the synoviolin gene deficient. The lacZ gene is introduced atthe translation starting position (ATG codon that is translated into thefirst methionine; indicated by “*”) of the mouse synoviolin genefragment, and a neomycin resistance (neo) gene is introduced as apositive selection marker gene. In addition, the diphtheria toxin A(DT-A) gene is also linked to form a negative selection marker.Individuals in which homologous recombination occurred lack theexpression of the synoviolin gene, but instead, β-galactosidase isexpressed and the expression from the promoter of the synoviolin genecan be detected by LacZ staining utilizing its enzyme activity (see FIG.22). The position of the probe used for Southern blot analysis (see FIG.20) in order to confirm the genotype is also illustrated.

FIG. 20 indicates a photograph showing the results of analysis of thegenotype of a synoviolin gene-deficient mouse. DNA is extracted fromtails of about two-week old mouse (wild type and hetero-deficient mouse)and from a 14.5 days post conception (dpc) fetus (homozygously-deficientmouse), and after digestion with PstI, Southern blotting was performedusing the probe shown in FIG. 19.

FIG. 21 indicates photographs showing the results of the Northern blotanalysis of a synoviolin gene-deficient mouse. mRNA was extracted from awild type (+/+), synoviolin gene heterozygously knock-out mouse (+/−)and homozygously knock-out mouse (−/−), and Northern blotting wasperformed using a synoviolin gene fragment as a probe (upper panel). Thelower panel illustrates EtBr staining of an agarose gel.

FIG. 22 indicates photographs showing the results of studying theSynoviolin expression location by LacZ staining. The 12.5 days postconception and 13.5 days post conception wild type andheterozygously-deficient mice were stained using LacZ. The expression ofSynoviolin in the embryonic stage was found to be strong in the parietalbone, limbs, ears and other locations where bone and cartilage form.

FIG. 23 indicates photographs showing the expression of Synoviolin inthe limb-formation stage. The expression of Synoviolin in thelimb-formation stage was found to be strong in the apical ectodermalridge (AER) in the same manner as the expression of FGF4, BMP2 and BMP4.

FIG. 24 indicates photographs showing the LacZ staining of a frozensection of a 13 days post conception limb bud of aheterozygously-deficient mouse. Staining was performed for 4 hours. Theblue of LacZ deeply stains undifferentiated mesenchymal cells (anlage ofbone and cartilage). Original magnification: ×40.

FIG. 25 indicates photographs showing the LacZ staining of a frozensection of a 13 days post conception limb bud of aheterozygously-deficient mouse. Staining was performed for 4 hours. Theblue of LacZ deeply stains undifferentiated mesenchymal cells (anlage ofbone and cartilage). Original magnification: ×200. A, B and C correspondto FIG. 24.

FIG. 26 indicates photographs showing the phenotype of synoviolin genehomozygously-deficient mice at 12.5 days and 13 days post conception.The synoviolin gene homozygously-deficient mice at 12.5 days and 13 dayspost conception exhibited a trend of a short length from the parietalregion to the buttocks, and a trend for the formation of the skull andlimbs to be premature. No marked differences in the phenotype were foundbetween the 13 days post conception heterozygously-deficient mouse andwild type mouse.

FIG. 27 indicates photographs showing the phenotype of a 14.5 days postconception synoviolin gene-deficient mouse. Limb bud abnormalities werefound in a 14.5 days post conception synoviolin genehomozygously-deficient mouse.

FIG. 28 indicates photographs showing the expression of LacZ in the hindlimbs of a 14.5 days post conception synoviolin genehomozygously-deficient mouse (reflecting the expression of Synoviolin).In the abnormal hind limbs of a homozygously-deficient mouse, LacZ wasfound to be expressed in sites where the AER and undifferentiatedmesenchymal cells are concentrated.

FIG. 29 indicates a photograph showing the phenotype of a 15.5 days postconception synoviolin gene-deficient mouse. Limb bud abnormalities andabnormal formation in the upper and lower jawbones and ears were foundin a homozygously-deficient mouse. No heartbeat was found and it was notalive.

FIG. 30 indicates photographs showing the skeleton of a 15.5 days postconception synoviolin gene-deficient mouse. Alcian blue and Alizarin redstaining are shown. Cartilage stained by Alcian blue and calcified bonestained by Alizarin red were not found in the synoviolinhomozygously-deficient mouse.

FIG. 31 indicates photographs showing a mouse arthritis model using ananti-collagen antibody cocktail in a synoviolin knock-out mouse. To thewild type mouse [373 (+/+)] and synoviolin heterozygously knock-outmouse [372 (−/+)], the anti-collagen antibody cocktail was administeredto elicit arthritis (+ in the figure). The non-administered wild typemouse (−) was also observed [371 (+/+)]. As a result, swelling andreddening of joints in both the front limbs and hind limbs were lesserin the synoviolin heterozygously knock-out mouse than in the wild type.To wit, the occurrence of arthritis was found to be weaker in thesynoviolin heterozygously knock-out mouse than the arthritis elicited inthe wild type mouse.

FIG. 32 indicates photographs showing the LacZ staining and Alcian bluestaining of primary cultured cells obtained from the limb buds of asynoviolin gene homozygously-deficient mouse (13 dpc fetus). The LacZpositive colony (to wit, the Synoviolin expressing cells) agreed withthe Alcian blue stain positive colony. This result suggests thatSynoviolin contributes to bone and cartilage differentiation. Passagenumber 1 (p1).

FIG. 33 indicates photographs showing LacZ staining of a primarycultured cell obtained from the limb bud of a 13 dpc mouse fetus. Cellsderived from a wild type mouse (+/+), and synoviolin gene heterozygously(−/+) and homozygously (−/−) deficient mice are shown. The expression ofLacZ is observed only in the synoviolin gene-deficient mouse (knock-inof the lacZ gene). Passage number 1 (p1).

FIG. 34 indicates photographs showing the LacZ staining and Alcian bluestaining of primary cultured cells obtained from the limb buds of asynoviolin gene heterozygously-deficient mouse (13 dpc fetus). The LacZpositive colony (to wit, the Synoviolin expressing cells) agreed withthe Alcian blue stain positive colony. Passage number 1 (p1).

FIG. 35 indicates photographs showing the LacZ staining and Alcian bluestaining of primary cultured cells obtained from the limb buds of a wildtype mouse (13 dpc fetus). Staining by LacZ is not observed. Passagenumber 1 (p1).

FIG. 36 indicates a photograph showing the LacZ staining of primarycultured cells obtained from the limb buds of a synoviolin geneheterozygously-deficient mouse (13 dpc fetus). LacZ staining (expressionof Synoviolin) is confirmed even in the typical binucleate cartilaginouscells (see the 200× image).

FIG. 37 indicates photographs showing the von Kossa staining of primarycultured cells obtained from the limb buds of fetal mice. Cells derivedfrom wild type (WT), synoviolin gene heterozygously (Hetero) andhomozygously (Homo) deficient mice are shown. A decrease in boneformation capacity is observed in the synoviolin gene-deficient mouse(Homo). Passage number 1 (p1).

FIG. 38 indicates photographs showing the LacZ staining of primarycultured cells (passage number 3; p3) obtained from the limb buds of asynoviolin gene homozygously-deficient fetal mouse. The culture wascontinued until it became subconfluent. After LacZ staining wasperformed (overnight), hematoxylin eosin (HE) staining was performed.

FIG. 39 indicates a diagram showing the results of a β-gal assay ofprimary cells of a synoviolin gene heterozygously knock-out mouse (lacZgene knock-in). The specimens were measured in triplicate and the meanand standard deviation are indicated.

FIG. 40 indicates a diagram showing the results of examining the effectsof various drugs on the synoviolin promoter activity by β-gal assay ofprimary cells of a synoviolin gene heterozygously knock-out mouse (lacZgene knock-in). The specimens were measured in triplicate and the meanand standard deviation are indicated.

FIG. 41 indicates a diagram showing the results of ELISA of mouse serumimmunized with Syno-P3. Serum obtained from three individuals (Nos. 1-3)was diluted in the indicated ratios and then ELISA was performed. Serumfrom a non-immunized mouse (“normal” in the diagram) was used as acontrol.

FIG. 42 indicates a diagram showing the results of ELISA of mouse serumimmunized with Syno-P2. Serum obtained from three individuals (Nos. 1-3)was diluted in the indicated ratios and then ELISA was performed. Serumfrom a non-immunized mouse (“normal” in the diagram) was used as acontrol.

FIG. 43 indicates a diagram showing the results of ELISA of mouse serumimmunized with Syno-P1. Serum obtained from three individuals (Nos. 1-3)was diluted in the indicated ratios and then ELISA was performed. Serumfrom a non-immunized mouse (“normal” in the diagram) was used as acontrol.

FIG. 44 indicates photographs showing the results of Western blotting(A) and fluorescent immunostaining (B) of synovial cells derived from RAand OA patients with anti-Synoviolin monoclonal antibodies.

FIG. 45 indicates photographs showing the results of immunostaining ofsynovial tissue derived from RA patients with anti-Synoviolin monoclonalantibodies. The hematoxylin eosin (HE) stain image is also shown.

FIG. 46 indicates photographs showing the auto-ubiquitination activityof Synoviolin. FLAG-Synoviolin was reacted in the presence ofGST-HA-ubiquitin, ATP, E1 and E2, and the ubiquitination of Synoviolinwas detected with Anti-FLAG antibodies and Anti-HA antibodies. CE: cellextract. IP: immune precipitate.

The following examples are offered to illustrate but not to limit theinvention.

EXAMPLE 1 Preparation of Anti-Synovial Cell Anti-Serum

Anti-synovial cell anti-serum was obtained using, as an immunogen,synovial cells prepared by the following procedure. Synovial tissueextracted by synovectomy from ten rheumatoid arthritis (RA) patients waswashed in phosphate buffered saline (PBS) in a sterile state. The washedtissue was cut to a size of approximately 5 mm square and 0.25%trypsin/PBS digestion was performed at 37° C. for 20 minutes. Excesstissue lumps were removed from the digested synovial tissue and thecells thus obtained were suspended in Dulbecco's modified Eagle's mediumcontaining 10% fetal calf serum (Virology (1959) 8:396) (10% FCS-DMEM)and cultured for 24 hours in a sterilized cell culture Petri dish under5% CO₂ at 37° C. The culture supernatant was discarded, washing wasperformed using 10% FCS-DMEM, and the non-adhering cells were removed toobtain rheumatism patient-derived synovial cells as cells adhered to thePetri dish (The Journal of Clinical Investigation (1993) 92:186). Thecultured cells were used as a pool and used in experiments as thefollowing synovial cells derived from RA patients.

Patient-derived synovial cells (1×10⁵) were suspended in 20 mL of 10%FCS-DMEM and cultured in a 76 cm² culture flask. The culture medium waschanged every 3 days and the culture surface was filled with cells aftertwo weeks, at which time the culture medium was removed and 7 mL each of0.05% EDTA/PBS and 0.1% trypsin/PBS were added to detach and recover thecells. The recovered cells were washed in PBS to remove the culturemedium components, and suspended in 1 mL of PBS to form an immunogen.

This immunogen was used within 2 hours after preparation to immunize onerabbit by intravenous injection into the ear. Immunization was performed6 times in total at one-week intervals. At the time of the sixthimmunization, when several mL of blood drawn from the ear of the rabbitwas tested for anti-serum, it was found by the fluorescent antibodymethod that the anti-serum reacted with the synovial cells of rheumatismpatients. One week after the sixth immunization procedure, a catheterwas used to draw as much blood as possible from the heart. This bloodwas kept overnight at 4° C. to coagulate and the serum was separated. Asa preservative, 0.1% sodium azide was added to the serum and the serumwas stored at 4° C. as anti-synovial cell anti-serum.

EXAMPLE 2 Gene Cloning of an Antigen (Synoviolin) Recognized byAnti-Synovial Cell Anti-Serum

The acid guanidine/phenol chloroform method was used to extract thetotal RNA from the synovial cells of ten RA patients obtained in Example1, and poly T beads were used to purify the mRNA (AnalyticalBiochemistry, 162, 159, 1987). The λZAP vector (Stratagene) was used toprepare a cDNA library of RA patient synovial cells by the ordinarymethod. A picoBlue immunoscreening kit (Stratagene) was used to performimmunoscreening with the anti-synovial cell anti-serum of Example 1above (FIG. 1). The positive clone (phage) thus obtained was convertedto a plasmid pBluescript II SK (+) with a helper phage. The nucleotidesequence of the DNA inserted into the pBluescript II SK (+) wasdetermined with an ABI PRISM 377 DNA Sequencer (PERKIN ELMER) usingM13PrimerM4 and M13PrimerRV (Takara) based on the dye terminator method(Proc. Natl. Acad. Sci. USA. (1977) 74:5463). The nucleotide sequencewas determined from the 3′ end of the gene (named “synoviolin”) thatencodes the antigen recognized by the above anti-synovial cellanti-serum, and a 2990 bp nucleotide sequence including a poly(A)⁺ chainwas clarified (SEQ ID NO: 1, No. 42-3031). Using this nucleotidesequence, a 3031 bp nucleotide sequence including the coding region offull-length synoviolin, a portion of the 5′-non-coding region and apoly(A)⁺ chain (SEQ ID NO: 1) was determined from the synovial cell cDNAlibrary by the 5′-RACE (Rapid Amplification of cDNA Ends) method (Proc.Natl. Acad. Sci. USA. (1988) 85:8998-9002). As a result of performing ahomology search in GenBank, this nucleotide sequence was found to be anew gene, with no similar sequence being reported.

EXAMPLE 3 Expression of Partial Synoviolin Recombinant Protein in E.coli

From a cDNA clone obtained by immunoscreening using anti-synovial cellanti-serum, cDNA that encodes a portion of Synoviolin (1799 bp; SEQ IDNO: 1, No. 1233-3031) was treated with the restriction enzymes EcoRI andXhoI and extracted. The cDNA which has a sequence recognized byEcoRI/XhoI at its ends was inserted into the glutathione S-transferase(GST) fusion protein expression vector pGEX-5X-3 and subcloning wasperformed. pGEX-5X-3 into which a portion of the synoviolin cDNA wasinserted was introduced into the BL21 E. coli strain by 45-second heatshock at 42° C. to obtain BL21/synoviolin-GST gene/pGEX-5X-3. This BL21was cultured in an LB medium containing 0.1 mg/mL ampicillin, 0.1 mMisopropylthio-β-D-galactoside (IPTG) was added, and it was cultured foran additional 2 hours at 37° C. to induce the expression of theaforementioned fusion protein. After the BL21 recovered bycentrifugation was washed in PBS, the BL21 was digested with 1 mg/mLlysozyme and solubilized with 0.1% Triton X-100. The BL21-derivedprotein suspension containing solubilized GST fusion protein was appliedto Glutathione Sepharose 4B (GS4B) and then washed with PBS, and 50 mMreduced form of glutathione/PBS was used to purify the desiredGST-partial Synoviolin fusion protein.

EXAMPLE 4 Expression of Full-Length Synoviolin Recombinant Protein in E.coli

Synoviolin cDNA (syno-HAHA) comprising the cDNA (1851 bp; SEQ ID NO: 1,No. 60-1910) that encodes Synoviolin obtained in Example 2 to which twomolecules of an influenza hemagglutinin (HA)-tag were added at the3′-end was inserted into the glutathione S-transferase (GST) fusionprotein expression vector pGEX-5X-1 and subcloning was performed.pGEX-5X-1 into which the syno-HAHA gene was inserted was introduced intothe BL21 E. coli strain by 45-second heat shock at 42° C. to obtainBL21/syno-HAHA/pGEX-5X-1. This BL21 was cultured in an LB mediumcontaining 0.1 mg/ml ampicillin, 0.1 mM isopropylthio-β-D-galactoside(IPTG) was added, and it was cultured for an additional 3 hours at 30°C. to induce the expression of the Synoviolin protein with GST fused tothe N terminus and HA fused to the C terminus (GST-Synoviolin-HAHA).After the BL21 recovered by centrifugation was washed in PBS, the BL21was digested with 1 mg/ml lysozyme and solubilized with 0.1% TritonX-100. The BL21-derived protein suspension containing solubilizedGST-Synoviolin-HAHA protein was applied to Glutathione Sepharose 4B(GS4B) and then washed with PBS, and 50 mM reduced form ofglutathione/Tris-HCl (pH 8.0) was used to purify the desiredGST-Synoviolin-HAHA protein.

Confirmation of expression was performed by 200 times and 2000 timesdiluting the fractions eluted with 50 mM reduced form of glutathione, bytreating them with 25 mM Tris-HCl (pH 6.8), 0.25% sodium dodecyl sulfate(SDS), 0.05% mercaptoethanol and 0.1% glycerol, and then by applyingthem to 8% SDS polyacrylamide electrophoresis (SDS-PAGE). AfterSDS-PAGE, GST-Synoviolin-HAHA protein was transferred to a nylonmembrane by electroblotting. This nylon membrane underwent blocking for60 minutes at room temperature in PBS containing 5% skim milk, and thenunderwent immune reaction for 60 minutes at room temperature withanti-HA monoclonal antibodies (Boehringer Mannheim) diluted 400 timeswith PBS containing 0.5% skim milk. After the reaction, it was washedwith 0.1% Tween 20/PBS, subjected to an immune reaction for 60 minutesat room temperature with horseradish peroxidase (HRP) labeled mouse IgGantibodies as the secondary antibodies, and washed with 0.1% Tween20/PBS, and the target antigen was detected by detecting HRP activity.The detection of HRP activity was performed using an ECL kit (Amersham)(Clinical Chemistry, 25, p. 1531, 1979). The results are shown in FIG.2. From the molecular weight size of the aforementionedGST-Synoviolin-HAHA fusion protein, the molecular weight of theSynoviolin protein is estimated to be approximately 80 kDa.

EXAMPLE 5 In Vitro Expression of Full-Length Synoviolin RecombinantProtein

The end of the synoviolin gene (SEQ ID NO: 1) was modified with therestriction enzyme EcoRI and inserted into the pBluescript II KS vector(syno/pBluescript). Thereafter, syno/pBluescript (1 μg) and TNT-coupledTranslation System (Promega) were used with in vitro translation toexpress the Synoviolin protein in vitro as an [³⁵S]-labeled protein. The[³⁵S]-labeled Synoviolin protein was applied to 10% SDS-PAGE and itsradioactivity was detected with an image analyzer (BAS2000, Fujix). Theresults are shown in FIG. 3. The molecular weight according to SDS-PAGEof the Synoviolin protein translated in vitro from the synoviolin genewas found to be approximately 80 kDa.

EXAMPLE 6 Confirmation of Expression of the Synoviolin Gene by NorthernBlotting

mRNA was obtained by ordinary methods from RA patient-derived synovialcells obtained in Example 1, the A549 cell line, Jurkat cell line andthe HeLa cell line. 1 μg of this mRNA was separated by 1% agarose gelelectrophoresis and transferred to a nylon membrane by contact blotting.The nylon membrane was treated for 2 hours at 80° C., andprehybridization was performed for 2 hours at 42° C. in Denhardt'ssolution. Next, using ³²P radiolabeled synoviolin cDNA (1799 bp; SEQ IDNO: 1, No. 1233-3031) as a probe, hybridization was performed for 12hours at 42° C. After the reaction, the nylon membrane was washed in 300mM NaCl and 30 mM sodium citrate, and then 15 mM NaCl and 1.5 mM sodiumcitrate were used to perform washing again at 50° C. The desired mRNAwas detected by exposure to X-ray film. The autoradiograph obtained as aresult is shown in FIG. 4. The synoviolin gene was found to be expressedstrongly in RA patient-derived synovial cells.

EXAMPLE 7 Confirmation of Expression of Synoviolin in Various Cells byWestern Blotting

The state of expression of Synoviolin was confirmed by Western blottingusing the following cells as specimens.

RA patient-derived synovial cells prepared in Example 1

Human umbilical vein endothelial cells (HUVEC)

HEK (human embryonic kidney)-293T

GST-partial Synoviolin fusion protein prepared in Example 3 (positivecontrol)

First, the various cells used as specimens were solubilized in 1% NP-40to prepare cell lysates. Each of the cell lysates was treated with 25 mMTris-HCl (pH 6.8), 0.25% sodium dodecyl sulfate (SDS), 0.05%mercaptoethanol and 0.1% glycerol, and then separated with 8% SDSpolyacrylamide electrophoresis (SDS-PAGE). After SDS-PAGE, proteinderived from the various cells was transferred to a nitrocellulose (NC)membrane by electroblotting. On this NC membrane, anti-synovial cellanti-serum was diluted 1000 times with Tris buffered saline (TBS)containing 2.0 mg/mL GST-partial Synoviolin fusion protein and 5% skimmilk, and subjected to immune reaction for 60 minutes at roomtemperature. In addition, as negative controls, an experiment whereinthe same antibody solution is reacted with the NC membrane, and anexperiment wherein the GST-partial Synoviolin fusion protein in theantibody solution is replaced with GST alone were performed at the sametime. After the reaction, the NC membrane was washed with 0.1% Tween20/TBS, subjected to an immune reaction for 60 minutes at roomtemperature with horseradish peroxidase (HRP) labeled anti-rabbit IgGantibodies as the secondary antibodies, and washed with 0.1% Tween20/TBS, and the target antigen was detected by detecting HRP activity.The detection of HRP activity was performed using an ECL kit (Amersham)(Clinical Chemistry, 25, p. 1531, 1979). The results are shown in FIG.5.

GST-partial Synoviolin blocked the immune reaction of the anti-synovialcell anti-serum to the 220 kDa protein that was detected in RApatient-derived synovial cells in the control experiments (FIG. 5;+GST), and not detected in the HUVEC and HEK-293T cells, and partiallyblocked its immune reaction to the approximately 140 kDa protein andapproximately 185 kDa protein (FIG. 5; +GST-partial Synoviolin).

The reactivity observed in bands other than 220-kDa band is presumed tobe fibronectin (molecular weight: approximately 240 kDa) or subunits oflaminin (molecular weight: approximately 200 kDa) determined by theirreactivity to other antibodies. Based on the results of theseexperiments, the molecular weight of Synoviolin is presumed to beapproximately 220 kDa. However, the molecular weight of Synoviolin asconfirmed in Example 5 is approximately 80 kDa. From the differencebetween the two, it is conceivable that Synoviolin has an multimericstructure that is not dissociated in SDS.

EXAMPLE 8 Confirmation of Expression of Synoviolin Protein in RAPatient-Derived Synovial Cells by Western Blotting

The RA patient-derived synovial cells prepared in Example 1 weresolubilized in 1% NP-40 to prepare cell extract fractions. This synovialcell extract was treated with 25 mM Tris-HCl (pH 6.8), 0.25% sodiumdodecyl sulfate (SDS), 0.05% mercaptoethanol and 0.1% glycerol, and thenseparated by 8% SDS polyacrylamide electrophoresis (SDS-PAGE). AfterSDS-PAGE, the synovial cell-derived protein was transferred to anitrocellulose (NC) membrane by electroblotting. On this NC membrane,anti-synovial cell anti-serum (in the figure, after immunization)obtained by immunizing RA patient-derived synovial cells was diluted1000 times with Tris buffered saline (TBS) containing 5% skim milk,allowed to undergo blocking for 1 hour at room temperature with Trisbuffered saline (TBS) containing 5% skim milk and subjected to immunereaction for 1 hour at room temperature. At the same time, serum drawnfrom a rabbit prior to the immunization of the rabbit with synovialcells (pre-immune) was used as a negative control. After reaction, theNC membrane was washed with 0.1% Tween 20/TBS, subjected to an immunereaction for 1 hour at room temperature with horseradish peroxidase(HRP) labeled anti-rabbit IgG antibodies as the secondary antibodies,and washed with 0.1% Tween 20/TBS, and the target antigen was detectedby detecting HRP activity. The detection of HRP activity was performedusing an ECL kit (Amersham) (Clinical Chemistry, 25, p. 1531, 1979). Theresults are shown in FIG. 6.

EXAMPLE 9 Confirmation of Expression of Synoviolin in Various Cells andSynovial Tissue by Immunostaining

Immunostaining was performed by fixing synovial cells upon glass slidesby the ordinary method, and immunostaining was performed using theanti-synovial cell anti-serum of Example 1. A sample subjected to30-minute blocking with 1% bovine serum albumin (BSA) was allowed toundergo immune reaction for 60 minutes at room temperature withanti-synovial cell anti-serum diluted 100 times with 1% BSA. Inaddition, along with observation with anti-serum, experiments were alsoperformed using anti-synovial cell antibodies purified from thisanti-serum. Purified anti-synovial cell antibodies were prepared byimmunoaffinity purification using GST-partial Synoviolin fusion proteinas a ligand. The ligand used was a fusion protein expressed after theGST-fusion protein expression vector pGEX-5X-3 containing the 1799 bpsynoviolin gene up to No. 1233-3031 of SEQ ID NO: 1 was transformed intoBL21. A Glutathione Sepharose column was produced by the method of thePharmacia Corp, to prepare a GST-partial syno-GS column. As a controlfor the case where purified anti-synovial cell antibodies were used, ananti-GST antibody obtained by the immunoaffinity purification ofanti-serum in the same manner where GST was used as the ligand was used.

After reaction, the sample was washed with PBS and then allowed toundergo immune reaction using fluorescein isothiocyanate-labeledanti-rabbit IgG antibodies as the secondary antibody. Confirmation ofthe antigen in the immune reaction with anti-synovial cell anti-serumwas performed with a confocal laser microscope. The results are shown inFIG. 7. This anti-serum exhibited a strong immune reaction with RApatient-derived synovial cells, and it was confirmed that this immunereaction was blocked by the GST-partial Synoviolin fusion proteinprepared in Example 3 (FIG. 7, top). Moreover, it was confirmed that theimmune reaction became even stronger and positive for purifiedanti-synovial cell antibodies prepared from this anti-serum (FIG. 7,bottom).

The staining of RA patient-derived synovial tissue was performed byfixing synovial tissue upon glass slides by the ordinary method. Asample subjected to 30-minute blocking with 1% BSA was allowed toundergo immune reaction for 60 minutes at room temperature withanti-synovial cell anti-serum diluted 100 times with 1% BSA. Afterreaction, the sample was washed with PBS and then allowed to undergoimmune reaction using HRP-labeled anti-rabbit IgG antibodies as thesecondary antibody. Confirmation of the antigen in the immune reactionwith anti-synovial cell anti-serum was performed by the coloring of3,3′-diaminobenzidine tetrahydrochloride based on HRP activity. In thesame manner as in the aforementioned Western blotting, GST-partialSynoviolin fusion protein was used to perform an anti-synovial cellanti-serum absorption test with respect to synovial tissue staining.Tissue staining was performed by adding 2.0 mg/mL of GST-partialSynoviolin fusion protein or GST (2.0 mg/mL) to anti-synovial cellanti-serum. The results are shown in FIG. 8. The staining of synovialtissue with anti-synovial cell anti-serum seen in the control was foundto be weakened by GST-partial Synoviolin fusion protein (FIG. 8). Inaddition, the immunostaining of synovial tissue using the aforementionedpurified antibodies from GST-partial Syno-GS was found to reactpositively in comparison to the antibodies obtained from GST-GS (FIG.9).

Based on the results of Western blotting (Example 8) and immunostaining,it was confirmed that the Synoviolin protein recognized by anti-synovialcell anti-serum was expressed in RA patient-derived synovial cells andsynovial tissue.

EXAMPLE 10 Presence of Anti-Synoviolin Antibodies in Serum of RAPatients

The present inventors attempted to detect anti-Synoviolin antibodies inthe serum of RA patients by Western blotting using GST-partialSynoviolin fusion protein as the antigen. Using the same procedure as inExample 7, first, GST-partial Synoviolin fusion protein (100 ng/lane)was electrophoresed by SDS-PAGE and transferred to an NC membrane. Asthe primary antibodies, RA patient serum (5 cases) was diluted 1000times with Tris buffered saline (TBS), and allowed to undergo immunereaction for 60 minutes at room temperature with the NC membrane ontowhich GST-partial Synoviolin fusion protein was transferred. The NCmembrane was washed with 0.1% Tween 20/TBS, subjected to an immunereaction for 60 minutes at room temperature with HRP-labeled anti-humanIgG antibodies as the secondary antibodies, and washed with 0.1% Tween20/TBS, and the human IgG that reacted with the target antigen wasdetected by detecting HRP activity. The detection of HRP activity wasperformed in the same manner as in Example 7. The results are shown inFIG. 10. Anti-IgG antibodies against GST-partial Synoviolin fusionprotein was found in the serum of RA-patients (five of five) (FIG. 10).On the other hand, antibodies that recognize GST-partial Synoviolin werenot found in serum derived from osteoarthritis (OA) patients and normalhuman serum.

EXAMPLE 11 Identification of Synoviolin Ligand by Screening anExpression Library

Screening for the Synoviolin ligand was performed using the cDNAexpression library derived from RA patient synovial cells prepared inExample 2 (Tadaomi Takenawa, Toshiki Watanabe, eds., Baiomanyuaru UPShirīzu “Tampakushitsu no Bunshikan Sōgosayō Jikken Hō” [Bio-Manual UPSeries “Protein Intermolecular Interaction Experimental Method”], pp.66-67, Yodosha Co., Ltd.; Kaelin, W. G. et al., Cell 70, 351-364, 1992;Skolnik, E. Y. et al., Cell 65, 83-90, 1991; Sambrook, J. et al.,Molecular Cloning, a laboratory manual second edition, Cold SpringHarbor Laboratory Press 12.16-12.20, 1989). The library phage wasinoculated into E. coli (XL1-Blue MRF′) by incubation for 20 minutes at37° C., and spread upon a plate after mixing with Top agarose. Afterculturing for 3.5 hours at 42° C., a nitrocellulose membrane soaked in10 mM IPTG and dried was placed upon a plate and culturing was performedfor an additional 3.5 hours at 37° C. After the membrane was recovered,it was washed five times for 5 minutes in a washing buffer [10 mMTris-HCl (pH 8.0), 0.5% skim milk, 0.1% Triton X-100, 150 mM NaCl, 1 mMEDTA, 5 mM MgCl₂, 1 mM DTT, protease inhibitor (complete, BoehringerMannheim)] and soaked for 1 hour in a blocking buffer [10 mM Tris-HCl(pH 8.0), 5% skim milk, 0.1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 5 mMMgCl₂, 1 mM DTT, 5% glycerol, protease inhibitor (complete, BoehringerMannheim)]. After 5-minute washing was performed five times with thewashing buffer, incubation was performed after adding, as a probe(approximately 10⁶ cpm/ml), GST-Synoviolin (the GST-partial Synoviolinfusion protein purified in Example 3) that was ³²P-labeled with proteinkinase A. Washing was performed repeatedly while changing the washingbuffer until the count per membrane became approximately 1 kcpm, andthen the signal was detected by autoradiography. As a result, a clonebound to Synoviolin was obtained. This clone was named Synoviolin ligand(SL).

Regarding the cDNA of SL, the nucleotide sequence was determined for 100bp near its 5′ end and 100 bp near its 3′ end. Upon performing adatabase search based on the nucleotide sequence information thusobtained, the sequence in the 100 bp portion at the ends was found to bethe same as that of a known gene called S1-5 [Lecka-Czernik, B. et al.,Molecular and Cellular Biology, 15, 120-128, 1995; accession numberU03877 (cDNA), AAA65590 (protein), also called “EFEMP1”: Stone, E. M. etal., Nature Genetics 22, 199-202, 1999; accession number Q12805(protein)]. The sizes of both genes are roughly the same, and the sizesof their translation products are roughly the same, suggesting that theyare the same protein.

EXAMPLE 12 Expression of the SL Gene by Northern Blotting

mRNA was extracted from various cells in the same manner as in Example6, and Northern blotting was performed using the SL cDNA obtained inExample 11 as a probe. The cells used are those given below. The RApatient-derived synovial cells were found to exhibit overexpression ofthe SL gene (FIG. 11).

HEK-293T

RA patient-derived synovial cells prepared in Example 1

A549

HeLa

EXAMPLE 13 Binding of Synoviolin to SL

The SL cDNA was inserted into a pGEX vector in the same manner as inExample 3 to prepare GST-SL fusion protein, and the GST-SL (500 ng) wasapplied to 10% SDS-PAGE along with GST (1 μg) as a control. AfterSDS-PAGE, it was transferred to a nylon membrane by electroblotting.This nylon membrane was denatured for 1 hour at room temperature with 50mM Tris-HCl (pH 8.0) containing 6M guanidine hydrochloride and 5 mM2-mercaptoethanol, and regenerated overnight at 4° C. in 50 mM Tris-HCl(pH 8.0) containing 5 mM 2-mercaptoethanol and 0.05% Tween 20. Theregenerated nylon membrane was treated with blocking buffer [10 mMTris-HCl (pH 8.0), 5% skim milk, 0.1% Triton X-100, 150 mM NaCl, 1 mMEDTA, 5 mM MgCl₂, 1 mM DTT, 5% glycerol, protease inhibitor (complete,Boehringer Mannheim)] and washed in blocking buffer (same as above,except for 0.5% skim milk). Thereafter, the TNT-coupled TranslationSystem (Promega) and pcDNA3-HA-synoviolin-HAHA (SEQ ID NO: 1 synoviolincDNA 1851 bp; synoviolin cDNA with an HA-tag added at 60-1910 insertedinto the expression vector pcDNA3) were used to perform in vitrotranslation, [³⁵S]-labeled HA-Synoviolin-HAHA fusion protein([³⁵S]HA-Synoviolin-HAHA) was used as a probe, and the GST-SL and GSTupon the nylon membrane were allowed to react for 2 hours at roomtemperature. This nylon membrane was washed in 10 mM Tris-HCl (pH 8.0),0.5% skim milk, 0.1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 5 mM MgCl₂, 1mM DTT, and protease inhibitor (complete, Boehringer Mannheim), and itsradioactivity was detected with an image analyzer (BAS2000, Fujix).Binding between the GST-SL fusion protein and [³⁵S]HA-Synoviolin-HAHAtransferred to the nylon membrane was observed. In addition, binding wasnot observed in the control of GST and [³⁵S]HA-Synoviolin-HAHA (FIG.12). From these results, Synoviolin and SL are presumed to bind byprotein interaction.

In addition, in Example 14, results are obtained that suggest thathyperplasia of synovial cells is blocked through Synoviolin-basedneutralization of Synoviolin ligand in a culture. Based on theseresults, mutants of SL that have a structure corresponding to theSynoviolin binding sites of SL are thought to possibly have the actionof suppressing the hyperplasia of synovial cells by antagonisticblocking action on the binding of Synoviolin to SL. Moreover, Synoviolinmutants that have a structure corresponding to the SL binding sites ofSynoviolin are also expected to have antagonistic blocking action in thesame manner as SL mutants.

EXAMPLE 14 MTT Assay

The RA patient-derived synovial cells prepared in Example 1 were used toprepare 96 well plates so that there were 5×10³ cells/well, and GST orGST-partial Synoviolin was added to the cell supernatant so that thefinal concentration became 0.01 to 1 μM. After three days of culture,3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)/PBS was added to the cell supernatant, and it was cultured for 3hours under conditions of 37° C. and 5% CO₂. After culturing, the cellsupernatant was removed, crystals of MTT formazan were dissolved withdimethyl sulfoxide, and absorbance measurement was performed (Journal ofImmunological Methods, 65, 55, 1983). Under conditions of 10%-FCS/DMEM,37° C., 5% CO₂, the RA patient-derived synovial cell hyperplasia wassignificantly suppressed by GST-partial Synoviolin (1 μM) (FIG. 13).

EXAMPLE 15 Preparation of a Synoviolin Gene-Introduced Mouse

A vector for expressing the synoviolin gene was constructed by linking aFlag tag to the N terminus of the DNA that encodes the Synoviolinprotein and linking a poly(A) signal downstream of the 3′ side. Thevector is constructed based on pCAGGS (Niwa, H., et al., Gene (1991)108:193-199) with a β-actin promoter as the promoter, and with humancytomegalovirus immediate early enhancer as the enhancer (FIG. 14).

The vector for expressing the synoviolin gene was injected into a mouseegg cell by microinjection using a microscopic glass pipette connectedto a manipulator under a microscope. DNA was injected into the malepronucleus of a fertilized egg and the injected manipulated egg wastransferred to the fallopian tubes of a female mouse (recipient mouse)in which false pregnancy was induced by breeding with a vasectomizedmale mouse. Mouse pups were obtained through natural parturition orcesarean section 19 days after transfer. In the case of a cesareansection, the mouse pups were nursed by a separately prepared femalemouse as a foster mother. DNA was taken from each tail of the newbornmice and PCR was used to confirm that it carries the transgene.

As a result, marked swelling of the joints was observed in Synoviolinoverexpression mice. The rate of onset of arthropathy in synoviolingene-expressed mice was found to be 33% (10 out of 30 individuals).Accordingly, the swelling of joints is thought to be not a natural-onsetmouse deformation (the rate of onset of hydrocephaly in the C57B6 mouseis less than 1%) but rather due to the contribution of the Synoviolinmolecule. A photograph taken by soft X-ray photography of the left hindlimb of a Synoviolin overexpression mouse is shown (FIG. 15).

EXAMPLE 16 Histological Study of Joints

The present inventors performed a histological study of the toe jointsof a synoviolin gene-expressed mouse (1 individual). Hematoxylin eosin(HE) staining of tissue sections of toe joint portions was performed.The hematoxylin eosin staining was performed according to known methods.

As a result of HE staining, bone disruption accompanying marked synovialhyperplasia and abnormal bone formation were found in portions thatexhibited arthropathy (FIG. 16). On the other hand, the aforementionedfindings were not observed in the normal toe joints of gene-expressedmice used as a control (FIG. 17, top).

In addition, as a result of performing immunostaining with anti-Flagantibodies in the toe joints, the expression of Synoviolin was found inthe synovial tissues and cartilaginous cells that exhibited hyperplasiain synoviolin gene-expressed mice (FIG. 18), but the aforementionedfindings were not observed in the normal toe joints of gene-expressedmice (FIG. 17, bottom).

EXAMPLE 17 Preparation of a Knock-Out Mouse

The lacZ gene was inserted in the translation starting position of themouse synoviolin gene fragment (ATG codon that is translated into thefirst methionine) to construct a targeting vector. As the marker gene, aneomycin resistance (neo) gene was inserted and the diphtheria toxin A(DT-A) gene was also linked to be able to exclude cell lines whereinnon-homologous recombination occurs (FIG. 19).

This targeting vector was transferred into a mouse ES cell TT-2 byelectroporation, and cell lines in which homologous recombinationoccurred were selected. The cells thus obtained were injected into amouse blastocyst or eight-cell stage embryo and either directlytransplanted to the fallopian tubes of a surrogate mother ortransplanted to the uterus of a surrogate mother after being culturedfor one day to develop into a blastocyst. Thereafter, a knock-out mousewas prepared by the same method as in the preparation of a transgenicanimal. The heterozygously mutated mice (F1) thus obtained were bred toeach other to obtain heterozygously and homozygously mutated mice. Inthe mutated mice thus obtained, the LacZ protein (β-galactosidase) isexpressed instead of Synoviolin in tissues where Synoviolin should beexpressed.

The genotype was confirmed by Southern blot analysis. Regarding the wildtype mice (14 individuals) and synoviolin heterozygously knock-out mice(32 individuals), DNA was extracted from a point roughly 3 mm from thetip of the tail of the mouse at age of about 2 weeks after birth. Withthe synoviolin homozygously knock-out mice, samples were taken under astereomicroscope from the tail and upper and lower limbs of 14.5 dayspost conception mice, and DNA was extracted. The DNA thus obtained wassubjected to digestion of the DNA with the restriction enzyme PstI andwas used. The results of analysis are shown in FIG. 20. Bands weredetected at 6.5 kbp in the wild type, at 8.5 kbp in the homozygouslymutated mice, and at both positions in the heterozygously mutated mice.

The expression of the synoviolin gene was confirmed by Northernblotting. mRNA was extracted from wild type, heterozygously knock-outmouse and homozygously knock-out mouse individuals (whole embryo at 12.5days post conception), and electrophoresis was performed with 20 μg ineach lane of 1.2% agarose gel. As a result, synoviolin mRNA was notdetected in homozygously knock-out mouse (−/−) individuals, while theexpression of mRNA in heterozygously knock-out mouse (+/−) individualswas observed to be weaker than that of wild type (+/+) individuals (FIG.21).

EXAMPLE 18 Study of Synoviolin Expression Sites

The present inventors used LacZ staining to study the Synoviolinexpression sites in the mutated mouse individuals obtained in Example17. To wit, 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) was usedto color the entire embryo and examine the distribution of expression ofLacZ (as β-galactosidase activity). The number of embryos observed was32.

As a result, strong expression of LacZ was found in the parietal boneand limbs at 12.5 days post conception, and in the ears and limbs at13.5 days post conception (FIG. 22). All of these were sites where boneor cartilage is formed. Moreover, as a result of performing LacZstaining and HE staining of limb tissue sections during the limbformation stage, strong expression was observed in the apical ectodermalridge (AER) and the anlage of cartilage and bone (or cartilage and bone)(FIG. 23).

Moreover, 13-day post conception limb buds were excised and frozensections were prepared. Then, hematoxylin eosin (HE) or LacZ stainingwas performed. Specifically, the frozen sections were washed three timesfor 5 minutes in PBS (−), and glass slides carrying the sections weresoaked in X-gal staining solution [X-gal (20 mg/ml) 1.25 ml, HEPES (1M)2.2 ml, potassium ferricyanide solution (100 mM) 1.5 ml, NaCl (5M) 150μL, MgCl₂ (1M) 65 μl, 10×PBS(−) 5 ml to which milli-Q water (Millipore)was added to reach 50 ml], thereby starting the reaction at 37° C. Afterstaining, dehydration was performed by an ethanol series and xylene andsealing was performed. As a result, the staining of the LacZ blue colorwas deep in the undifferentiated mesenchymal cells (the anlage of boneand cartilage) (FIGS. 24 and 25).

EXAMPLE 19 Study of Phenotypes

Moreover, the present inventors studied the phenotypes of the synoviolingene knock-out mouse.

At 12.5 days post conception, the homozygotes exhibited a trend of ashorter length from the parietal region to the buttocks than that of theheterozygotes, and a trend for the formation of the skull and limbs tobe premature, but at 13 days post conception, there was no markeddifference in the phenotype between the heterozygotes and wild types(FIG. 26). Besides, no births of homozygous mouse individuals werefound, and no live homozygous mouse embryos were found after at least 17days post conception. Accordingly, these were thought to be fetal deaths(Table 1).

TABLE 1 Number Wild Homozygote Age analyzed type Heterozygote (alive ordead) 12.5 dpc 10 0 8 2 (alive)   13 dpc 10 2 7 1 (alive) 14.5 dpc 6 2 31 (?) 15.5 dpc 6 0 5 1 (dead)   4 weeks old 46 14 32 0

At 14.5 days post conception, no marked difference was observed betweenheterozygotes and homozygotes in the length from the parietal region tothe buttocks. However, while the toes and joints were formed in theheterozygotes, they were not formed in the homozygotes and limbabnormalities were found (FIG. 27).

Moreover, as a result of performing LacZ and HE staining on the hindlimbs that exhibited abnormalities in the homozygotes, the expression ofLacZ which reflects the expression of Synoviolin was found in theconcentrated sites of the AER and undifferentiated mesenchymal cells(locations of future toe bone formation) (FIG. 28).

At 15.5 days post conception, the homozygotes were dead, exhibitingmorphological abnormalities in the limb buds, upper and lower jaws andears (FIG. 29). Moreover, cartilaginous tissue was stained by Alcianblue and bone (calcified) tissue was stained by Alizarin red. To wit,the epidermis, dermis and contents of a mouse were removed, soaked infixer (ethanol:hydrogen peroxide=9:1), dehydrated in an alcohol series,and then stained with Alizarin red and Alcian blue, and the tissue wasmade transparent with an alkaline solution. After made transparent, itwas kept in a glycerin solution and staining was observed. As a result,no formation of cartilaginous tissue (stained blue) or bone (calcified)tissue (stained red) was found in the homozygote (FIG. 30).

From the aforementioned results, the synoviolin gene homozygouslyknock-out mouse was found to exhibit developmental abnormalities in itslimb buds in the fetal stage. In addition, the formation of cartilageand bone was not found, and Synoviolin was found to be expressed in thelocations of development of the limb buds, cartilage and bone.Accordingly, the contribution of the Synoviolin molecule to skeletalformation is strongly conceivable.

EXAMPLE 20 Administration of Cocktail for Arthritis to the SynoviolinGene Knock-Out Mouse

Collagen-induced arthritis (CIA) in the mouse is widely used as anarthritis model for human rheumatoid arthritis. An anti-collagenantibody cocktail was administered to the synoviolin gene knock-outmouse (heterozygote) prepared in Example 17 and to a wild-type mouse,and the arthritis elicited was observed. As a result, the elicitation ofarthritis in the synoviolin heterozygously knock-out mouse was found tobe weaker that that of the wild type (FIG. 31). These results alsosupport the fact that Synoviolin contributes to the induction ofarthritis in RA.

EXAMPLE 21 Analysis of a Primary Culture of Fetal Limb Bud Cells of aSynoviolin Gene Knock-Out Mouse

Among the cells obtained (by the explant method) from a knock-out (KO)mouse, LacZ staining, namely the expression of Synoviolin, was foundonly in the undifferentiated mesenchymal cells thought to be the anlageof cartilage, bone and limbs. In addition, in the primary culture offetal limb bud cells, the LacZ positive colony (to wit, the Synoviolinexpressing cells) agreed with the Alcian blue stain-positive colony, andmoreover, the staining of LacZ (as β-galactosidase activity) (expressionof Synoviolin) is observed in the typical binucleate cartilaginous cellsalso. This supports the fact that Synoviolin is involved in bone andcartilage differentiation. Moreover, by alkaline phosphatase staining,von Kossa staining or other methods, it was confirmed that the capacityto form bone and cartilage was delayed in homozygously knock-out derivedcells (FIGS. 32-38).

In van Kossa staining, after the cells were washed, the solution wassubstituted with silver nitrate solution (5% w/v). After the cells werelightly washed with distilled water, reduction and fixing were performedwith sodium thiosulfate solution (5% w/v). After washing,counterstaining was performed with Kernechtrot solution (0.1% w/vKernechtrot (Nuclear Fast Red), 5% w/v aluminum sulfate) (Masaji Seki,Soshiki Kensa Hō—Soshiki Kōzō to Kyokusho Kagaku—[Tissue Test Methods:Tissue Structure and Local Chemistry], 257-258, Kyorin-Shoin, 1961; L.Lison, Tadashi Imaizumi, trans., Histochimie et Cytochimie Animales:Principes et Mëthodes [Animal Histochemistry and Cytochemistry:Principles and Methods], 625-636, Hakusuisha Publishing Co. Ltd., 1962;Yutaka Sano, Soshikikagaku Kenkyū Hō—Riron to Jutsushiki [HistochemistryResearch Methods: Theory and Practice], 616-621, Nanzando Co., Ltd.,1965). The detection of alkaline phosphatase activity was performed withan alkaline phosphatase tissue staining kit (Sigma, Diagnostic Kits andReagents, alkaline phosphatase (AP), leukocyte, Cat. No. 86-R).

EXAMPLE 22 Test Compound Assay Using Synoviolin Gene Knock-OutMouse-Derived Cells

Primary cultured cells of a synoviolin gene heterozygously knock-outmouse (lacZ gene knock-in) were used to evaluate the effect of a testspecimen on the expression of the synoviolin gene by β-gal assay. Theprimary cultured cells of a synoviolin gene heterozygously knock-outmouse after 3 passages were seeded to 24 well plates at 0, 1×10³, 3×10³,1×10⁴, 3×10⁴ and 1×10⁵ cells per well, and cultured overnight inDulbecco's modified Eagle's medium (DMEM) containing 10% fetal calfserum. First, cell lysis solution (Promega) was added to the cellculture in the absence of stimulation in a quantity (100 μl/well)sufficient to cover the cell layers completely. Then, the culture plateswere moved to a shaking machine and shaken gently for 15 minutes at roomtemperature so that the cell layers were always soaked in lysissolution.

The β-galactosidase activity of the cells was measured in the followingmanner. To 20 μl of the obtained cell extract solution were added 1 μlof Mg solution (0.1M MgCl₂, 4.5M β-mercaptoethanol), 22 μl of ONPGsolution (o-nitrophenyl-β-D-galactopyranoside) (concentration of 4 mg/mlin a 0.1M phosphate buffer (pH 7.5)), and 57 μl of 0.1M phosphate buffer(pH 7.5), to give a total volume of 100 μl. With care not to allow it todry, incubation was performed for 6 hours at 37° C. The reaction washalted by adding 150 μl of 1M sodium carbonate solution (prepared bydissolving 21.2 g of Na₂CO₃ in H₂O, by adjusting to 200 ml and byfiltering with a 0.45 μm filter), and the β-galactosidase activity wasquantified by measuring the absorbance at 420 nm. The experiment wasperformed in triplicate. As a result, β-galactosidase activity wasdetected in the lacZ gene knock-in mouse cells depending on the numberof cells (FIG. 39). The present inventors confirmed that the evaluationof promoter activity (β-gal assay) is possible using β-galactosidaseactivity as an index.

Next, β-gal assay was performed in the same manner by adding variousdrugs to the primary cultured cells, and then the effects of the variousdrugs on synoviolin promoter activity were evaluated. As a negativecontrol, the same measurements were performed by adding the culturemedium only. The test drugs used were prednisolone (0.01-1 μM) and12-O-tetradecanoylphorbol 13-acetate (TPA; 0.001-0.1 μM). Prednisoloneis a steroidal anti-inflammatory drug, and TPA is a protein kinase Cactivator.

5×10⁴ primary culture cells were seeded in each well, and afterculturing overnight, the various drugs were added at the aforementionedconcentrations. After the drugs were added, the cells were cultured for72 hours and the β-galactosidase activity of each well was measured toevaluate the promoter activity. As a result, the synoviolin promoteractivity was found to be affected by these drugs in aconcentration-dependent manner (FIG. 40). To wit, this confirmed thatthe activity of the drug on the synoviolin promoter could be evaluatedwith the assay system based on the present invention. From the resultsabove, such an assay can be used to evaluate the effect of various drugson the synoviolin promoter activity, and thus, it is possible to screenfor compounds that promote or suppress the synoviolin promoter activity.

EXAMPLE 23 Preparation of Anti-Synoviolin Monoclonal Antibodies

Monoclonal antibodies to Synoviolin were prepared as follows. Aspeptides used for immunization, the following three peptides containingpartial amino acid sequences of human Synoviolin were synthesized. Theseamino acid sequences were selected from among the domains assumed tohave antigenicity.

Syno-P3 (SLALTGAVVAHAYYC/SEQ ID NO: 3),

Syno-P2 (TCRMDVLRASLPAQS/SEQ ID NO: 4), and

Syno-P1 (GAATTTAAGTSATAC/SEQ ID NO: 5).

Keyhole limpet hemocyanin (KLH) was conjugated to each of thesynthesized peptides via Cys within the amino acid sequence. 50 μg ofeach of the synthesized peptides conjugated to KLH was dissolved in 0.1ml of physiological saline solution, and 0.1 ml of Freund's completeadjuvant (FCA) was added to prepare an immunogen. Each immunogen (0.2ml) was injected hypodermically into the back of eight mice (BALB/cfemale, 5 weeks old), thus immunizing it. Immunization was performedonce every two weeks for four times in total, and immunization wasperformed one more time one week later. Eight days after the finalimmunization, blood was drawn from the heart to obtain 200 μl or moreserum. Spleen cells were taken from individuals in which an increase inthe antibody titer was confirmed by ELISA, and then cell fusion wasperformed.

FIGS. 41-43 show the results of measuring the antibody titer by ELISAfor the mouse serum of three individuals with respect to each of theimmunogens. Each serum sample was assayed in triplicate, and the mean isshown on the graph. Individuals in which the antibody titer increasedwere confirmed when any of the immunogens was used. Thus, it wasconfirmed that each of these immunogens was useful as an immunogen ofSynoviolin.

Myeloma cell line (P3U1) cells and mouse spleen cells were mixed in a1:10 ratio and cell fusion was performed in the presence of 50% PEG(PEG1540 of Wako Pure Chemical Industries, Ltd.). After fusion, 96 wellplates were seeded so that the spleen cell count became 5×10⁵/ml. Afterthe cells were cultured for 10-14 days in a HAT culture medium, cellgrowth was confirmed and the culture supernatant was tested. An ELISAplate on which the various synthesized peptides was fixed was used totest the culture supernatant. The testing procedures are as follows.After the culture supernatant was reacted with the ELISA plate,anti-mouse IgG goat-pox was used to select positive wells. The wells tobe used for cloning were selected and the cells of other positive wellswere frozen and stored.

Several days later, each strain was seeded over one 96-well plate at 100cells/plate (20 cells/ml), and cultured for 10-14 days. The colonieswere determined and testing of the culture supernatant was performed.Testing of the culture supernatant was performed by applying 50 μl ofsupernatant to the aforementioned antigen-fixed ELISA plates forscreening. Anti-mouse IgG goat-pox was used as the second antibody.After cultured, the selected colonies were recloned and cultured for10-14 days. Then, colony determination and testing of the culturesupernatant were performed in the same manner as given above. Wells wereselected according to mother strain, and selected clones were culturedin a 24-well plate. The supernatant was recovered, and clones werechecked. Then, the antibody subclass and antibody production weretested. As a result of the cloning, using Syno-P2 (SEQ ID NO: 4) as theimmunogen, the two clones 10 Db and 7 Bc were selected as the hybridomasthat produce monoclonal antibodies having the high affinity toSynoviolin.

EXAMPLE 24 Detection of Synoviolin in Patient Specimens UsingAnti-Synoviolin Monoclonal Antibodies

<1> Western Blotting of Patient-Derived Synovial Cells withAnti-Synoviolin Monoclonal Antibodies

Using the two types of anti-Synoviolin monoclonal antibodies (10 Db and7 Bc) that recognize Syno-P2 obtained in Example 23, proteins ofrheumatoid arthritis (RA) patient-derived synovial cells were separatedby SDS-PAGE and Western blotting was performed. The Western blottingprocedure was as recited in Example 8 except that the monoclonalantibodies 10 Db and 7 Bc of Example 23 were used as the antibodies andanti-mouse IgG sheep-HRP was used as the labeled antibody. As a control,osteoarthritis (OA) patient-derived synovial cells were also analyzed.As a result, a signal specific to RA patient-derived synovial cells wasdetected (FIG. 44A). It was confirmed that the monoclonal antibodiesobtained in Example 23 specifically recognized the synovial cells of RApatients. These monoclonal antibodies are useful in the detection of RA.

<2> Fluorescent Immunostaining of RA Patient-Derived Synovial Cells withAnti-Synoviolin Monoclonal Antibodies

The monoclonal antibody 10 Db was used to perform fluorescent immunecytochemical analysis of RA patient-derived synovial cells. Theimmunostaining procedure was as recited in Example 9 except that themonoclonal antibodies 10 Db of Example 23 were used as the antibodiesand anti-mouse IgG sheep-FITC was used as the labeled antibody. TheSynoviolin protein signal was detected strongly in RA patient-derivedsynovial cells, but it was not detected in the control wherein only thesecondary antibodies were reacted (FIG. 44B).

<3> Immunostaining of RA Patient-Derived Synovial Tissues withAnti-Synoviolin Monoclonal Antibodies

The monoclonal antibodies 10 Db and 7 Bc were used to performimmunostaining of synovial tissue sections taken from RA patients. Theimmunostaining procedure was as recited in Example 9 except that themonoclonal antibodies 10 Db and 7 Bc of Example 23 were used as theantibodies and anti-mouse IgG sheep-HRP was used as the labeledantibody. The Synoviolin protein signal was detected strongly in RApatient-derived synovial tissue (FIG. 45). A hyperplastic layer ofsynovial cells was observed by HE staining performed at the same time,and it was confirmed that the portion was stained by monoclonalantibodies. Based on these results, it was confirmed that the monoclonalantibodies of the present invention specifically recognized the synovialtissue of RA patients. As described above, RA testing and diagnosis canbe performed by detecting Synoviolin in patient specimens usingSynoviolin antibodies.

EXAMPLE 25 Detection of the Ubiquitin Ligase Activity of Synoviolin

E3 ubiquitin-protein ligase is known to undergo auto-ubiquitination(Hashizume R. et al., J. Biol. Chem. 276, 14537-14540, 2001). Thus, thepresent inventors studied whether Synoviolin has auto-ubiquitinationactivity or not. Plasmids comprising a FLAG-synoviolin gene insertedinto a pCAGGS vector were transfected into HEK-293 cells and the cellswere recovered 36 hours later. A cell extract was obtained with Buffer A[15 mM Tris-HCl pH 7.5, 0.5 M NaCl, 0.35% NP-40, 1 mM PMSF, 2 μg/mlaprotinin, 2 μg/ml leupeptin]. The cell extract was centrifuged in ahigh-speed centrifuge. To 0.6 ml of the supernatant, 3 μg of anti-FLAGantibodies and 7.5 μl of Protein A beads were added, andimmunoprecipitation was performed overnight. The beads were washed threetimes with Buffer A or Buffer A to which 0.1% SDS was added, and thenwashed two times with Buffer B [25 mM Tris-HCl pH 7.5, 50 mM NaCl, 0.01%Nonidet P-40, 10% glycerol, 1 mM EDTA]. Then, 30 μl of ubiquitin ligasereaction solution [50 mM Tris-HCl pH 7.4, 5 mM MgCl₂, 2 mM NaF, 10 nMokadaic acid, 2 mM ATP, 0.6 mM DTT, 1.5 μg GST-HA-ubiquitin, 40 ngyeast-derived E1, 0.3 μg UbcH5c(E2)] was added and it was allowed toreact for 30 minutes at 37° C. 30 μl of 2× Laemmli SDS-loading buffercontaining 0.1M DTT was added and boiled. Then, it was fractionated bySDS-PAGE and transferred to a nitrocellulose membrane. Anti-FLAGantibodies (SIGMA) and anti-HA antibodies (Roche Diagnostics) were usedas the primary antibodies. The detection of HRP activity was performedin the same manner as in Example 7. As controls, the extract from cellstransfected with the FLAG-synoviolin gene only (beforeimmunoprecipitation) and the solution obtained by performingimmunoprecipitation of the cell extract (i.e. FLAG-Synoviolin proteinand its immunocomplex only) were used. Moreover, reactions were alsoperformed without adding any one of GST-HA-ubiquitin, ATP, E1 and E2(when GST-HA-ubiquitin was not added, the reaction was performed usingGST). FIG. 46 shows the results of using Synoviolin immunopurified bywashing with 0.1% SDS-containing Buffer A. In the blotting with anti-HAantibodies, a band whose size was about 35 kDa larger than the molecularweight of Synoviolin (* in FIG. 46) was detected (the arrow in FIG. 46).This band was also observed in the blotting with the anti-FLAGantibodies, and is thought to be that of a protein in whichGST-HA-ubiquitin is fused to Synoviolin. Moreover, the reaction systemsdeficient in any one of ATP, E1 and E2 indicated that theauto-ubiquitination of Synoviolin did not occur. The same result wasobtained when the beads were washed with Buffer A only. From theseresults, it is clear that 1) E1- and E2-dependent ubiquitin ligaseactivity is present in Synoviolin-containing immune complexes, and fromthe results of immunopurification, 2) Synoviolin has E3ubiquitin-protein ligase activity.

INDUSTRIAL APPLICABILITY

The present invention provides the gene “synoviolin” which encodes anovel protein that contributes to the development of synovial membranesand to the development of bone, cartilage and limbs. The gene accordingto the present invention is involved in RA and antibodies to theproducts of this gene are produced in RA patients. The gene and proteinaccording to the present invention become new markers useful in thediagnosis of RA. The “synoviolin” according to the present invention isoverexpressed in the joint synovial cells of RA patients and contributesto the diagnosis of the disease RA and judgment of the effectiveness oftreatment by in situ hybridization and in situ PCR. Moreover, antibodiesto Synoviolin can be detected with a high frequency in the blood of RApatients. Specific diagnosis of RA is possible using this as a marker.The Synoviolin protein provided by the present invention, or partialpeptides thereof, are useful in the detection of antibodies toSynoviolin in the serum of patients.

In addition, Synoviolin is also expressed in undifferentiatedmesenchymal cells. If Synoviolin is used as a cell marker, then it ispossible to recover undifferentiated mesenchymal cells from fetal cellsor the like. Undifferentiated mesenchymal cells are cells that aredifferentiated into bone and cartilage and are expected to haveapplications in regenerative medicine. To wit, if undifferentiatedmesenchymal cells recovered using Synoviolin as a cell marker aredifferentiated in vitro or in vivo and the formation of bone orcartilage or reconstruction of joints is performed, it becomes possibleto reconstruct anew bones, cartilaginous tissue or joints that havesuffered injuries.

The Synoviolin and its ligand according to the present invention havebeen shown to have a close relationship with the hyperplasia of jointsynovial cells which is a major pathology of RA. Accordingly, theSynoviolin or its ligand provided by the present invention givesimportant knowledge in the development of RA treatment methods. Morespecifically, by performing the screening of compounds that are involvedin the binding between Synoviolin and its ligand, it is possible toproceed with the development of RA treatment techniques by a completelydifferent approach from that taken previously. Moreover, in thesynoviolin transgenic mouse, hyperplasia of the joint synovial membranesand swelling of toe joints accompanying arthritis occur with a highfrequency. The synoviolin transgenic animals provided by the presentinvention are extremely useful as a model of RA in the development oftreatment techniques and pharmaceuticals.

1. An isolated and purified protein comprising SEQ ID NO:2.
 2. Theprotein of claim 1 that is encoded by the polynucleotide of SEQ ID NO:1.